Effect of supplementation with brewer’s yeast hydrolysate on growth performance, nutrients digestibility, blood profiles and meat quality in growing to finishing pigs

Objective This study was aimed to investigate the effects of brewer’s yeast hydrolysate (YH) on growth performance, nutrients digestibility, blood profiles and meat quality of growing pigs. Methods A total of 200 growing pigs ([Landrace×Yorkshire]×Duroc) (initial body weight, 25.31±1.29 kg) were allotted to 5 treatments as follow: CON, basic diet; and YH treatment, CON+0.05%, 0.1%, 0.5%, and 1.0% of YH, respectively. Results On wk 11, 16 and overall phase, pigs fed YH diet showed a linear improvement in average daily body gain and gain/feed (p<0.05). The pigs that received YH linearly increased the digestibility of dry matter, nitrogen, and energy on wk 11 and 16. The concentration of serum urea nitrogen was linearly increased in YH treatments on wk 16. However, the carcass weight, back fat and lean muscle percentage of pigs receiving YH had no significant change. Besides, no difference was observed in creatinine and total protein in the blood among treatments. Conclusion The pigs fed a graded YH diet had improved growth performance and nutrient digestibility, meanwhile, the YH increased the serum urea nitrogen in the growing pigs.


INTRODUCTION
Over thousands of years, brewer yeast has been widely used for alcohol fermentation in the brewing process. Meanwhile, as eukaryote unicellular organisms, yeast has strong fermentation ability, which has a lower toxic potential with a high nutritional value [1][2][3]. Nowadays, yeast fermentation derivative products with bioactive properties including yeast cells and yeast extracts have been known to improve dietary palatability and fiber digestibility, prevent pathogen growth, produce antibacterial compounds and modulate the immune system [4][5][6][7].
Compared with yeast extracts, hydrolyzed yeast has a lower cost advantage by reducing production process without extraction. The methods of the manufacturing preparation of hydrolysates are divided into autolysis and enzymatic hydrolysis which were used to hydrolyze the yeast cells to release the nutrients. Results have indicated that enzymatic hydrolysis is the more effective method [8][9][10].
A downstream process of yeast extract production was developed from brewer's yeast cells, including various processing conditions as clarification, debittering, and the Maillard reaction. Some studies followed a process of manufacture where the yeast cells were washed, centrifuged and then heat-inactivated before drum drying after fermentation [11]. However,

The manufacture of a brewer's yeast hydrolysate
Brewer's yeast was inserted into the vessel with HCl (Cas No. 7647-001-0. Duksan corp., Seoul, Korea) and heated to the target temperature 105°C. When vessel temperature was 105°C, the sample was held for 4 h under the controlled temperature and cooled down to 45°C. After cooling, the samples were removed from the vessel, and passed through a mash filter (1.0×1.0 mm).
Brewer's YH bioprocesses were by polysaccharide culture from the STR Biotech company, Ltd (Chuncheon, Korea). Brewer' s yeast and L. edodes fungal mycelia were isolated from the mushroom fruitbody and cultured on yeast mold agar (Difco Laboratory, Detroit, MI, USA) and potato dextrose agar medium (PDA, Difco Laboratory, USA). The brewer's yeast with mycelia cultured on PDA media was inoculated in 50 mL of the liquid medium ( Table 1). The culture experiments were commenced in 250 mL Erlenmeyer flasks at 28°C for 5 d in a rotary shaker (120 rpm) and that were then used to seed the main liquid culture. The main liquid medium contained black rice bran (20 g/L) and dried soybean powder (2 g/L). The medium was then treated with amylase and protease at 60°C for 60 min for enzymatic digestion of particulate materials containing carbohydrate and protein. Subsequently, the culture mass was adjusted to pH 6.0 with brewer's YH, followed by sterilization in the autoclave. The experiment with the main liquid culture was started using a 5 L fermentor (working volume of 3 L) at 28°C and 150 rpm by inoculating with the inoculum (10%) of the pre-liquid cultured mycelia. After 7 days, the culture mass was ground in a colloid mill (model PUC60 Hankook Power Technology System, Seoul, Korea). The powder was treated with 0.1 M lactic acid for 60 min, followed by treatment with an enzyme mixture for cell wall lysis containing cellulase, hemicellulase, pectinase, glucanase, mannase, and arabinase at 50°C for 60 min.

Experimental design, animals, and diets
A total of 200 crossbred growing pigs ([Landrace×Yorkshire] ×Duroc) with an average initial body weight of 25.31±1.29 kg were used in feeding trial for 16 weeks. Pigs were randomly allotted to 5 experiment treatments. Dietary treatments include: CON, basic diet; YH0.05, CON+0.05% YH; YH0.1, CON+0.10% YH; YH0.5, CON+0.50% YH; and YH1, CON+ 1.0% YH. There were 8 replicates per treatment with 5 pigs (three gilts and two barrows) per replicate. All diets were provided in mash form and formulated to meet or exceed the NRC [13] recommendations for all nutrients ( Table 2) including growing pig phase (wk 1 to 6) and finishing pig phase (wk 6 to 16). All the pigs were housed in an environmentally controlled room with a slatted plastic floor. Throughout all the experimental period, each pen was equipped with a 1-sided self-feeder and a nipple drinker to allow the pig's ad libitum access to feed and water.

Sampling and measurements
Growth performance: Body weight and feed consumption were measured initially and wk 6, 11, and 16 to monitor the average daily gain (ADG), average daily feed intake (ADFI), and gain/feed (G/F) ratio.
Nutrients digestibility: Apparent total tract digestibility (ATTD) of dry matter (DM) and nitrogen (N) were determined by adding chromic oxide (0.2%) as an inert indicator in the diet. Pigs were fed diets mixed with chromic oxide one week before collecting samples at the last day of wk 6, 11, and 16. Fresh fecal grab samples collected from at least 2 pigs per pen by rectal massage were stored in a freezer at -20°C until analyzed. Before chemical analysis, the fecal samples were thawed and dried at 60°C for 72 h, after which they were finely ground to a size that could pass through a 1-mm screen. All feed and fecal samples were analyzed for DM and N following the procedures outlined by the AOAC [12]. Chromium was analyzed via UV absorption spectrophotometry (Shimadzu, UV-1201, Shimadzu, Kyoto, Japan) following the method described by Williams et al [14]. Energy (E) was determined by measuring heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA). Nitrogen content was determined (Method 920.40) [12] and crude protein was calculated as N×6.25. The ATTD was calculated using the following formula: Where N f = nutrient concentration in faces (% DM), N d = nutrient concentration in diet (% DM), C d = chromium concentration in diet (% DM) and C f = chromium dioxide concentration in faces (% DM).
Blood profiles and carcass grade X: At the end of the experi-ment (16 wk), blood samples were taken from each pig by Vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). Serum samples were isolated (centrifuged at 3,000×g for 15 min) 2 to 4 h after collection. An automatic blood analyzer (HITACHI 747, Kyoto, Japan) was used analyze blood urea nitrogen (BUN), total protein and creatinine in the serum samples. At the end of the experiment (16 wk) the pigs were slaughtered in a slaughterhouse and a report on carcass grade was received from the web site of Korea Institute for Animal Products Quality Evaluation.
Statistical processing: The data were analyzed using the general linear model procedure of SAS (SAS Institute, Inc., Cary, NC, USA, 1996) as a randomized complete block design. Pen served as the experimental unit. Orthogonal comparisons were conducted using polynomial regression to measure the linear, quadratic, and cubic effects of increasing the supplementation YH. Results were expressed as the least squares means and standard error of mean. Probability values less than 0.05 were considered significant.

Growth performance
The effects of YH on growth performance are shown in Table  3. On wk 11, 16, and overall period, the ADG linearly increased for pigs fed YH, meanwhile, the ADG of pigs cubically increased and G/F was linearly improved on wk 6 (p<0.05). Moreover, the G/F of pigs fed YH also linearly increased since they consumed YH at every stage of the experiment. There was no significant effect of increased content of ADFI during experiment phase.

Nutrient digestibility
The results of nutrient digestibility are shown in Table 4. On wk 11, only digestibility of DM (p = 0.002) and N (p = 0.030) were linearly increased in the response of increasing dietary concentrations of supplemental YH. On wk 16, there was a linear increased in DM (p = 0.004), N (p = 0.030), and E (p = 0.021) in the response to increasing dietary concentrations of supplemental YH. However, on wk 6, there were no significant effects on nutrient digestibility among treatments.

Meat quality
The results of carcass grading are shown in Table 5. There was no significant difference in meat quality among the treatments. Table 6 shows results of blood profile from growing-finishing pigs. There was no significant difference among the treatments in creatinine and total protein. However, the BUN of pigs linearly increased as the inclusion rate of YH increased (p = 0.049).

Growth performance
The yeast cell and extract has used in the livestock industry for many decades. Many previous studies demonstrated that yeast cells have a positive effect on growth performance of pigs. Li et al [15] reported that the live yeast (Saccharomyces cerevisiae) increased quadratically body weight gain and feed intake during a starter experiment with weaning pigs. Meanwhile, Håkenåsen [11] showed that the ADG was linearly  In the present study, the ADG and G/F were linearly increased in the overall period by diet with added graded YH (from 0.05% to 1%). Few papers about YH used in the diet were found which lead us to consider other yeast derivatives. Our results are consistent with Dvorak and Jacques [17] and Li et al [18] that a diet with added yeast extract has a positive effect on the body weight gain and growth efficiency in the pigs. Meanwhile, Zhao et al [19] found that the ADG and ADFI were increased in the pigs fed a nursery diet containing yeast extract. The reason ADFI was increased may be due to improving palatability in the dietary after adding yeast. A similar viewpoint that pigs receiving diets with higher levels of yeast had improved the feed intake [3,11]. Furthermore, some researchers thought that the yeast cells which were treated with multiple enzymes had a better flavor profile since the enzymes strongly affected the degree of hydrolysis and protein compositional characteristics [20].
According to Kemp and Kiser [21] glucan as the main component of the yeast cells, which has significant influence on growth performance of animals. The probable reason is that yeast cells produced and released various proteolytic, glycolytic, or lipolytic enzymes to digest organic matter, or absorbed amino acids and monosaccharides which has the effect of suppressing bacteria, thereby increasing growth performance.
A similar explanation is that growth performance was improved by assimilation of nutrients that are directly used for growth and because of β-glucan inhibits the production of cytokines, which can be produced by pathogenic microbial infections in the animal [22]. In addition, some papers found that a diet with added yeast had a significant influence on the bone growth, for example, tibial bone, bonefemur bone growths and growth plate (proximal epiphysis) in the rat [23]. The mechanism of the positive effects on animals produced by a diet with relevant yeast needs to be researched.

Digestibility nutrients
As described above, after treated by enzyme mixture, the mixture of yeast cells and culture increased not only feed intake but also digestibility of nutrients. As many studies have demonstrated the pig diet with added protease, cellulose or enzyme mixture could apparently enhance the digestibility of crude protein, energy, and dry mater, thereby improving the growth performance of pigs [24][25][26]. Those results are consistent with our research that the digestibility of DM and N were increased at wk 11 and the digestibility of E was increased at wk 16 by diet concentration of supplemental YH which contributed to the growth performance in pigs.
Our results are supported by Keimer et al [6] that the digestibility of CP and E tended to increase in 1.0% hydrolyzed yeast group. We speculate that the healthy morphology of the intestine was improved by diet with added YH. Håkenåsen [11] researched that fecal DM was linearly increased after adding levels of yeast, meanwhile, Keimer et al [6] showed  that weaned pigs had significantly increased villus height, villus/crypt ratio in jejunum, and reduced crypt depth in the colon when fed a diet with added 1% hydrolyzed yeast. Carlson [27] has more detailed observations that after being fed a yeast diet, nursery pigs had shorter crypt depths which means that fewer cells were migrating to the villus to ad digestion and absorption. However, different results have reported that short-term supplementation with dietary yeast [15,27] in the diet of weaned pigs had no influence on the ileal DM, CP, and crude fiber digestibility.
It is a hypothesized that the β-glucan may positively effect digestion and absorption rate by increasing the immune system and maintaining health in the state of infectious diseases. Hiss et al [22] reported that the nutrient utilization rate (digestibility of DM, E, CP, calcium, and phosphorous) of young piglets fed β-glucan was significantly improved. But it needs further research into how β-glucan directly affects the digestibility of nutrients.

Meat quality
That dietary supplementing with yeast could favorably improve the quality of edible meat from broilers has been proved by many trials. For example, edible meats from broiler chicks fed a diet containing enriched yeast (Saccharomyces cerevisiae) exhibited increased tenderness and increased water holding capacity [28,29]. Some results showed that dietary 0.3% yeast supplementation improved the antioxidant status in muscle. Meanwhile, increased levels of yeast decreased the drip loss and the concentration of thiobarbituric acid reactive substances in the muscle and meat [30]. Another point is that the addition of yeast to the diet improves the quality of meat by decreasing fatty acids. In a study of diet added 0.5% yeast extract to the broiler, the oxidative fat was decreased [30]. Onifade et al [31] researched that a diet supplement of Saccharomyces cerevisiae to broilers decreased abdominal fat mass. We searched the literature and found that the probable reason yeast had significant influenced on the birds' meat was due to the yeast improving the oxidative stability of meat.
However, the data was limited for the effect of yeast cells or yeast metabolic products on meat quality in pigs. In the present paper, carcass weight, back fat and lean muscle percentage of pigs were not significantly different. As opposed to a paper that carcass weight and breast muscles were increased by a diet containing Saccharomyces cerevisiae yeast cells [32]. Therefore, further research is required to investigate the effect of yeast on the quality of meat in pigs.

Blood profile
The response of blood profile in the diet supplemented with graded of YH is shown in Table 6 where serum urea nitrogen was increased, while creatinine and total protein were not influenced in the pigs. We speculated that the diet pro-moted N absorption after added YH and for this is reason the digestibility of N was increased in our experiment. However, studies about blood profiles effects of brewer's yeast are limited. A similar result that the plasma of serum urea nitrogen was increased by diet contained hydrolyzed yeast in weaned pigs has been reported [33]. It has been discovered that diet with added yeast extract could decrease the content of cholesterol in the blood. One study was reported that β-glucan from Saccharomyces cerevisiae fed to high cholesterol group of men decreased total cholesterol in the blood [34].
Otherwise, we searched the literature for the influence on other blood profiles in animals fed yeast. We found that a diet supplement of YH could help animals increase growth hormones (GHs) and immune responses. For example, Kim et al [23] demonstrated that the diet with a maximum of 1.0% of yeast enhanced GH in the rats. That GH was increased by adding yeast in the diet is a probable reason why the pigs could improve the growth performance after fed a yeast diet in the present experiment. Elsewhere, some papers directly demonstrated that yeast metabolites could promote improving immune responses. Waititu et al [33] found that yeast added multi-enzyme mixture could reduce ileal interferon-γ and interleukin-10 expression after challenged Escherichia coli lipopolysaccharide in weaned pigs. Jeney and Anderson [35] demonstrated that yeast extract promoted specific and nonspecific immune responses by enhancing the acid phosphates activity of peritoneal macrophages, neutrophil activity and produce oxidative radicals. On blood profiles, there are needs to further study various effects of hydrolyzed brewer's yeast.

CONCLUSION
The effect of increasing supplementation with brewer's YH was to improve the growth performance with body weight and feed efficiency, besides, the apparent digestibility of nutrients in some phases was significantly increased, and the content of blood urea nitrogen was improved in the growing pigs. However, there was no significant effect on creatinine, total protein and carcass grade in growing to finishing pig.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.