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Animal Science Journal (2014) ••, ••–••

doi: 10.1111/asj.12335

ORIGINAL ARTICLE Comparative efficacy of up to 50% partial fish meal replacement with fermented soybean meal or enzymatically prepared soybean meal on growth performance, nutrient digestibility and fecal microflora in weaned pigs Jinsuk JEONG and In Ho KIM Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam, South Korea

ABSTRACT This study was conducted to determine the comparative efficacy of partial fish meal (FM) replacement (up to 50%) with fermented soybean meal (FSBM; SoELAB, PepSoyGen and Soytide) or enzymatically prepared SBM (HP 300) on growth performance, nutrient digestibility and fecal microflora in weaned pigs. A total of 100 weaned pigs (body weight 6.59 ± 0.29 kg) were used in experimental feeding trials, lasting for up to 6 weeks, and were randomly allotted to five groups with four block replicates of five pigs per pen serving as one block. Dietary treatments were as follows: (i) 100% FM, (ii) 50% FM + 50% SoELAB-54, (iii) 50% FM + 50% PepSoyGen, (iv) 50% FM + 50% Soytide and (v) 50% FM + 50% HP 300. Concerning growth performance, none of the treated SBM preparations demonstrated any significantly different effect compared with FM treatment. With respect to nutrient digestibility, SoELAB and HP 300 treatments demonstrated no significant difference compared with FM treatment. Lastly, none of the SBM preparations demonstrated any significant differences in animal fecal score and all of the differentially treated SBM increased fecal Lactobacillus counts, while maintaining similar Escherichia coli counts compared with FM treatment.

Key words: enzymatically prepared soybean meal, fermented soybean meal, fish meal, growth performance, nutrient digestibility.

INTRODUCTION Currently, fish meal (FM) is commonly used as the prime protein source in weaned pig diets due to the high digestibility of nutrients, favorable amino acid (AA) composition, and lack of anti-nutritional factors (ANF) which can reduce nutrient availability (Li et al. 1991; Choct et al. 2010) and negatively affect growth performance of young pigs (Kim et al. 2000a,b, 2010; Jones et al. 2010). ANF includes antigens, lectins, trypsin inhibitors (TI) and oligosaccharides (Kim & Easter 2001; Pierce et al. 2005). However, FM’s high cost as an ingredient has necessitated the identification of alternative cheaper protein sources for diets fed to weaned pigs. Interestingly, soybean meal (SBM) is an important protein source fed to adult pigs because of its excellent balance of indispensable AA (Baker 2000; Wang et al. 2011) and low fiber concentration, which results in a greater concentration of metabolic energy than in © 2014 Japanese Society of Animal Science

other oilseed meals (Stein et al. 2008). Compared with the majority of other protein sources, SBM has a relatively high concentration of lysine and tryptophan, which is positive since these two limiting AA are relatively low in most cereal grains and in corn in particular (Sharda et al. 1976). However, SBM contains ANF and therefore its use as the sole source of AA in weaned pig diets is not recommended (Dunsford et al. 1989). On the other hand, fermented and enzymatically prepared SBM have been used to ameliorate the negative effects of the weaning lag (Yang et al. 2007; Min et al. 2009). Hence, treated SBM may serve as a

Correspondence: In Ho Kim, Department of Animal Resource and Science, Dankook University, Cheonan, Choongnam 330-714, South Korea. (Email: inhokim@ dankook.ac.kr) Received 10 July 2014, accepted for publication 28 August 2014.

2 J. JEONG and I. H. KIM

viable partial replacement for FM in weaned pig diets. Treated SBM has reduced amounts of ANF and a greater concentration of crude protein (CP) and AA than untreated SBM (Hong et al. 2004; CervantesPahm & Stein 2010). Therefore, the objective of the current study was to determine the comparative efficacy of FM replacement, up to 50% of FM portion, with popular commercially available fermented and/or enzymatically prepared SBM sources (SoELAB-54, PepSoyGen, Soytide and HP 300) in weaned pigs.

MATERIALS AND METHODS Animals and diets A total of 100 weaned pigs ((Yorkshire × Landrace) × Duroc) with an average initial body weight (BW) of 6.59 ± 0.29 kg were randomly allotted to one of five treatment groups, with each group consisting of 20 animals, with four replicate blocks of five pigs per pen, in accordance with a randomized complete block design. All of the pigs were housed in an environmentally controlled nursery facility with slatted plastic flooring and a mechanical ventilation system. Each

pen was equipped with a one-sided, stainless-steel self-feeder and a nipple drinker which allowed pigs unlimited access to feed and water throughout the experiment. Diets were formulated to meet or exceed the nutrient requirements by the NRC (2012) for pigs. Feed ingredients and calculated nutrient composition of the basal diet are shown in Tables 1 and 2, respectively. Five dietary treatments were prepared by mixing FM with different protein sources, and were as follows: (i) FM: 100% FM (prepared from FF-Skagen (Skagen, Denmark)); (ii) SoELAB: 50% FM + 50% SoELAB-54 (FEEDUP Co., Nonsan, Korea); (iii) PepSoyGen: 50% FM + 50% PepSoyGen (NutraFerm, North Sioux City, SD, USA); (iv) Soytide: 50% FM + 50% Soytide (CJ Cheiljedang Bio, Seoul, Korea); and (v) HP 300: 50% FM + 50% HP 300 (Hamlet protein, Horsens, Denmark). SoELAB-54 is produced by fermentation of SBM in the presence of Enterococcus faecium SLB120. PepSoyGen is produced by fermentation of SBM in the presence of Apergillus oryzae and Bacillus subtillis. Soytide is produced by fermentation of SBM in the presence of B. subtillis. Lastly, HP 300 utilizes a proprietary enzymatic preparation to reduce all ANF in SBM to a perfectly safe level for young animals. Diets were fed for 1 week (Phase I) and 2 weeks (Phase II) in mash form, and then each group was switched onto common commercial diet as crumble for 3 weeks (Phase III).

Table 1 Ingredient composition of experimental diets – Phase 1 (as-fed basis)

Items

Treatment† FM

Ingredient, % Corn Soybean meal (48% crude protein) Fish meal SoeLAB PepSoyGen Soytide HP 300 Soybean oil Whey dried Lactose Dicalcium phosphate Limestone Sugar L-Lysine DL-Methionine L-Threonine Vitamin premix‡ Mineral premix§ Spray dried animal plasma Calculated compositions Metabolic energy, kcal/g Crude protein Lysine Methionine Cysteine Methionine + Cysteine

SoELAB

PepSoyGen

Soytide

HP 300

27.68 17.50

27.35 17.70

26.89 17.90

27.39 17.60

26.94 17.75

5.00

2.50 2.50

2.50

2.50

2.50

2.50 2.50 4.50 15.00 15.50 1.68 1.20 5.00 0.300 0.240 0.10 0.10 0.20 6.00

4.33 15.00 15.50 1.75 1.30 5.00 0.375 0.295 0.10 0.10 0.20 6.00

4.60 15.00 15.50 1.75 1.30 5.00 0.375 0.290 0.10 0.10 0.20 6.00

4.40 15.00 15.50 1.75 1.30 5.00 0.375 0.290 0.10 0.10 0.20 6.00

2.50 4.70 15.00 15.50 1.75 1.30 5.00 0.375 0.290 0.10 0.10 0.20 6.00

3569 20.97 1.507 0.557 0.37 0.929

3557 20.91 1.508 0.552 0.37 0.925

3560 20.93 1.505 0.558 0.37 0.933

3565 20.92 1.505 0.559 0.37 0.934

3564 20.90 1.506 0.558 0.37 0.932

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. ‡Provided per kilogram of complete diet: vitamin A, 10 000 IU; vitamin D3, 2000 IU; vitamin E, 48 IU; vitamin K3, 1.5 mg; riboflavin, 6 mg; niacin, 40 mg; d-pantothenic, 17 mg; biotin, 0.2 mg; folic acid, 2 mg; choline, 166 mg; vitamin B6, 2 mg; and vitamin B12, 28 μg. §Provided per kilogram of complete diet: Fe (as FeSO4·7H2O), 90 mg; Copper (as CuSO4·5H2O), 15 mg; Zinc (as ZnSO4), 50 mg; Mn (as MnO2), 54 mg; I (as KI), 0.99 mg; and Se (as Na2SeO3·5H2O), 0.25 mg.

© 2014 Japanese Society of Animal Science

Animal Science Journal (2014) ••, ••–••

FERMENTED SOYBEAN MEAL IN PIGS

Table 2

3

Ingredient composition of experimental diets – Phase 2 (as-fed basis)

Items

Treatment† FM

Ingredient, % Corn Soybean meal (48% CP) Fish meal (LT) SoeLAB-54 PepSoyGen Soytide HP 300 Soybean oil Whey dried Lactose Dicalcium phosphate Limestone Sugar L-Lysine DL-Methionine L-Threonine Vitamin premix‡ Mineral premix§ Spray dried animal plasma Calculated compositions Metabolic energy, kcal/g Crude protein Lysine Methionine Cysteine Methionine+Cysteine

44.80 21.50 3.00

SoELAB 44.30 21.80 1.50 1.50

PepSoyGen 44.05 21.80 1.50

Soytide 44.40 21.70 1.50

HP 300 44.05 21.80 1.50

1.50 1.50 3.30 10.00 8.00 1.52 0.90 3.00 0.360 0.22 0.10 0.10 0.20 3.00

3.25 10.00 8.00 1.62 0.99 3.00 0.395 0.25 0.10 0.10 0.20 3.00

3.50 10.00 8.00 1.62 0.99 3.00 0.395 0.25 0.10 0.10 0.20 3.00

3.25 10.00 8.00 1.62 0.99 3.00 0.395 0.25 0.10 0.10 0.20 3.00

1.50 3.50 10.00 8.00 1.62 0.99 3.00 0.395 0.25 0.10 0.10 0.20 3.00

3509 20.05 1.655 0.604 0.42 1.022

3503 20.03 1.661 0.601 0.42 1.079

3507 20.03 1.663 0.606 0.42 1.030

3504 20.06 1.659 0.607 0.42 1.030

3506 20.05 1.661 0.605 0.42 1.027

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. ‡Provided per kilogram of complete diet: vitamin A, 10 000 IU; vitamin D3, 2000 IU; vitamin E, 48 IU; vitamin K3, 1.5 mg; riboflavin, 6 mg; niacin, 40 mg; d-pantothenic, 17 mg; biotin, 0.2 mg; folic acid, 2 mg; choline, 166 mg; vitamin B6, 2 mg; and vitamin B12, 28 μg. §Provided per kilogram of complete diet: Fe (as FeSO4·7H2O), 90 mg; Copper (as CuSO4·5H2O), 15 mg; Zinc (as ZnSO4), 50 mg; Mn (as MnO2), 54 mg; I (as KI), 0.99 mg; and Se (as Na2SeO3·5H2O), 0.25 mg.

The levels of each protein source added were 5% (Phase I), 3% (Phase II) and 0% (Phase III) of diet. The experiment was carried out for a total of 6 weeks, during which experimental samples were noted at the end of each phase of experimental feeding. The institutional Animal Care and Use Committee of Dankook University (Anseodong, Cheonan, Choongnam, Korea) reviewed and approved the protocols for all experiments.

Nutrient and ANF analysis of SBM preparations All SBM preparations were analyzed for dry matter (DM) (Method 934.01; AOAC 2000), crude fat (ether extract) (Method 920.39; AOAC 2000) and ash (Method 942.05; AOAC 2000). Chromium was analyzed using UV absorption spectrophotometry (Shimadzu UV-1201; Shimadzu, Kyoto, Japan), according to the method described by Williams et al. (1962). Gross energy (GE) was determined by measuring heat of combustion in the samples, using a bomb calorimeter (Parr 6100; Parr Instrument Co., Moline, IL, USA). Individual AA composition was measured using an AA analyzer (Beckman 6300; Beckman Coulter, Inc., Fullerton, CA, USA), after 24 h hydrolysis with HCl (Spackman et al. 1958). Nitrogen (N) content was determined (Method 920.40; AOAC 2000) and CP was calculated as N × 6.25. Animal Science Journal (2014) ••, ••–••

Differentially treated SBM samples were analyzed for trypsin inhibitors (TI) (Method 71–10; AACC 1976). TI concentration was analyzed using UV absorption spectrophotometry (Shimadzu UV1201; Shimadzu), according to the method described by Chang and Tsen (1976). For raffinose and stachyose measurements, crude fat was removed from 10 g of milled sample by 50 mL of diethyl ether and then saccharides were extracted using 100 mL of 80% ethanol for 2 h according to the soxhlet extraction process. After the solution was filtered through a cellulose filter (0.45 μm pore size; Whatman, Dassel, Germany), the eluent was evaporated thoroughly in a 40°C water bath and then dissolved in 20 mL distilled water. The dissolved sample was drawn through the activated carbon and C18 SPE cartridges (Varian, Palo Alto, CA, USA) at a flow rate of 5-10 m/min to remove the color and non-polar contaminators. A 20 μL aliquot of filtered solution was injected into an high performance liquid chomatograph (HPLC) (LCS1121/RI detector; Sykam, Damascus, Syria) fitted with a Shodex Asahipak NH2P- 50 4E column (Shoko, Tokyo, Japan) (4.6 mm internal diameter × 250 mm) and eluted with 25/75 (v/v) of H2O/CH3CN at a flow rate of 1 mL/min at 30°C. The retention times and concentrations of raffinose and stachyose (Fluka Chemicals, Gilingham, UK) were compared and determined from standard solutions of these sugars. © 2014 Japanese Society of Animal Science

4 J. JEONG and I. H. KIM

Growth performance and nutrient digestibility analysis Average daily gain (ADG) and feed intake (FI) were recorded on initial day, week 1, week 3 and end of the feeding trial, which was then used to calculate gain/feed (G/F) ratio after correcting for mortality. Individual pig BW and FI per pen was determined at the end of each phase to calculate BW gain (BWG), FI and G/F. From days 0 to 7 (Phase I) and from days 15 to 21 (Phase II), chromic oxide (0.2%) was added to diets as an indigestible marker for determination of apparent total tract digestibility (ATTD) of dry matter (DM), N and GE, respectively. Fresh fecal samples were obtained by massaging the rectum from at least two pigs per pen at days 7 and 21. Fecal samples from the same pen were pooled and mixed immediately, after which samples were stored in a freezer at −20°C until analysis. Before chemical analysis, feces 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 sieve.

ferment for 7 days at room temperature (25°C). The concentrations of gas were determined on day 7 during the fermentation period. A gas sampling pump (Model GV-100; GASTEC, Ayase, Japan) was utilized for gas detection (Gastec detector tube No. 3La for ammonia; No. 4LK for hydrogen sulfide; No. 70 for mercaptan; GASTEC). Prior to measurements, slurry samples were manually shaken for approximately 30 s for homogenization and disruption of any crust formation on the surface of the slurry sample. The adhesive plasters were punctured and 100 mL of headspace air was sampled approximately 2 cm above the slurry surface. Two samples from each pen were measured and then the mean was calculated.

Intestinal microflora analysis

Blood samples (10 mL) were randomly collected, via anterior vena cava puncture, from eight pigs in each treatment at the end of Phase I and Phase II experiments. Blood samples were collected into 5 mL vacuum tubes (Becton Dickinson Vacutainer Systems; BD Co., Franklin Lakes, NJ, USA) and tripotassium ethylenediaminetetraacetic acid (K3EDTA) vacuum tubes (BD Co.) and stored at 4°C. Samples for blood analysis were then centrifuged at 3000 × g for 15 min, and then separated and stored at 4°C. Blood urea nitrogen (BUN), creatine and glucose concentrates were analyzed using an automatic biochemical blood analyzer (HITACHI 747; HITACHI, Tokyo, Japan). White blood cells (WBC), red blood cells (RBC) and lymphocyte percentages of the whole blood samples were analyzed using an automatic blood analyzer (ADVIA 120; Bayer, Tarrytown, NY, USA).

Fresh feces samples were collected on weeks 1 and 3 by massaging the anus and were put into Qorpak glass containers (118 mL) under CO2, sealed, and placed on ice until transported to the laboratory for enumeration of microbial populations. These samples were assessed for populations of Lactobacilli and Escherichia coli. One gram of composite excreta sample from each pen was diluted with 9 mL of 1% peptone broth (BD Co.) and then homogenized. Viable counts of bacteria in the fecal samples were then conducted by plating serial dilutions (10−3 to 10−7) onto de MannRogosa-Sharpe (MRS) agar plates (Difco Laboratories, Detroit, MI, USA) and MacConkey agar plates (Difco Laboratories) supplemented with glacial acetic acid (1 mL/L) and mupirocin (100 mg/L) extracted from antimicrobial discs (Oxoid, Nepean, Canada) to isolate the Lactobacilli and E. coli, respectively. The MRS agar plates and MacConkey agar plates were then incubated for 38 h at 37°C under anaerobic conditions. The microflora colonies were counted immediately after removal from the incubator, and concentration of microflora was finally expressed as log10 colony-forming units per gram of intestinal content.

Excreta gas emission analysis

Statistical analysis

Fecal samples were scored according to the following index used by Murray et al. (1998: 1 = hard, dry pellet (small and hard mass); 2 = hard, formed, dry stool (remains firm and soft); 3 = soft, formed, moist (softer stool that retains shape); 4 = soft, unformed (stool assumes shape of container and pudding-like); and 5 = watery (liquid that can be poured). Fresh excreta samples were collected from each pen after week 3 of the experiment and then mixed well for each respective pen. Analysis was then conducted using excreta samples (150 g feces and 150 g of urine were well mixed; 1:1 on a wet weight basis) obtained from each pen which were stored in 2.6 L sealed plastic containers. Each container had a small hole in the middle of one side wall, which was sealed with adhesive plaster. The samples were permitted to

All experimental data were analyzed as a randomized complete block design, with one pen representing an experimental block unit. Data were analyzed by the GLM procedure of SAS (2001). The model was a one-way analysis of variance, and the analysis of variance employed Tukey’s test in order to indicate significant differences (P < 0.05) among means.

Blood profile analysis

RESULTS ANF and nutrient composition analysis of SBM preparations The concentrations of ANF from differentially treated SBM are shown in Table 3. Decreasing concentrations

Table 3 Anti-nutritional factors in fermented soybean meal and enzymatically prepared soybean meal in weaned pigs

Items

Treatment† SoELAB

Trypsin inhibitor, mg/g Raffinose, % Stachyose, %

2.25 ± 0.08 0.08 ± 0.01c 0.31 ± 0.01c

b

PepSoyGen

Soytide

3.02 ± 0.16 0.18 ± 0.03a 0.54 ± 0.03a

2.14 ± 0.03 0.13 ± 0.01b 0.46 ± 0.01b

a

HP 300 bc

1.77 ± 0.07c 0.12 ± 0.01b 0.43 ± 0.01b

†SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. a,b,cMeans ± SD in the same row with different superscripts differ (P < 0.05).

© 2014 Japanese Society of Animal Science

Animal Science Journal (2014) ••, ••–••

FERMENTED SOYBEAN MEAL IN PIGS

Table 4

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Nutrient composition of fermented soybean meal and enzymatically prepared soybean meal (as-fed basis)

Items

Ingredient, % DM CP Ether extract Fiber Ash Indispensable AA, % Arg His Ile Leu Lys Met Phe Thr Try Val Total TIAA/CP Dispensable AA, % Ala Asp Cys Glu Gly Pro Ser Tyr Total TDAA/CP

Treatment† FM

SoELAB

PepSoyGen

Soytide

HP 300

91.28 67.40 8.81 0.27 12.67

92.55 54.52 0.89 3.78 6.04

91.41 52.29 1.01 3.99 7.63

95.29 55.69 0.98 3.46 7.84

91.55 53.46 1.24 4.10 7.29

4.20 1.74 3.16 5.47 5.82 1.78 3.08 3.02 0.46 3.55 32.28 47.89

3.52 1.39 2.61 4.31 3.35 0.37 2.88 2.11 0.48 2.62 23.64 43.36

3.55 1.32 2.55 4.28 3.06 0.59 2.81 2.21 0.50 2.72 23.59 45.11

3.57 1.43 2.63 4.48 3.36 0.44 2.99 2.19 0.60 2.67 24.36 43.74

3.90 1.42 2.51 4.33 3.46 0.58 2.86 2.26 0.57 2.61 24.50 45.83

4.34 6.20 0.77 9.36 4.10 2.27 2.69 2.03 31.76 47.12

2.33 5.82 0.64 9.86 2.25 2.21 2.60 1.61 27.32 50.11

2.42 6.11 0.85 10.05 2.32 2.04 2.71 1.63 28.13 53.80

2.41 6.03 0.64 10.11 2.30 2.20 2.62 1.64 27.95 50.19

2.46 6.17 0.84 10.51 2.33 2.27 2.76 1.65 28.99 54.23

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%; DM, dry matter; CP, crude protein; TIAA, total indespensible amino acids; TDAA, true digestible amino acids.

of ANF, according to treatment, was in the following order: (i) TI: PepSoyGen>SoELAB ≈ Soytide>HP 300; (ii) raffinose: PepSoyGen>Soytide ≈ HP 300>SoELAB; and (iii) stachyose: PepSoyGen>Soytide ≈ HP 300>SoELAB, respectively. Overall, HP 300 contains the lowest amount of ANF sampled out of all SBM preparations. Nutrient composition analyses of differentially treated SBM preparation are shown in Table 4.

Growth performance The effect of the differentially treated SBM preparations (SoELAB, PepSoyGen, Soytide and HP 300) on growth performance are shown in Table 5. The SoELAB and HP 300 treatments showed no significant differences in BW compared with FM treatment (P > 0.05); however, supplementation of PepSoyGen and Soytide reduced BW at weeks 3 and 6, and week 6, respectively (P < 0.05). During Phase I (days 0 to 7), no effects of treated SBM treatment were observed on ADG, average daily feed intake (ADFI), and G/F compared with FM treatment (P > 0.05), respectively. Animal Science Journal (2014) ••, ••–••

During Phase II (days 8 to 21) and Phase III (days 22 to 42), no effects of treated SBM treatment were observed on ADG and G/F (P > 0.05); however, supplementation of SoELAB, PepSoyGen, Soytide, and HP 300 reduced ADFI compared with FM (P < 0.05), respectively. Throughout the whole period (days 0 to 42), the supplementation of PepSoyGen and Soytide reduced ADG; additionally, all of the SBM treatments reduced ADFI compared with FM treatment (P < 0.05), respectively. As a consequence, supplementation of PepSoyGen reduced G/F compared with FM (P < 0.05).

Nutrient digestibility The effects of the differentially treated SBM on nutrient digestibility are shown in Table 6. SoELAB treatment and HP 300 treatment showed no significant difference in ATTD of DM, N and GE compared with FM treatment, respectively (P > 0.05). PepSoyGen treatment reduced ATTD of N at week 1 and Soytide treatment increased ATTD of GE at week 1 compared with FM treatment, respectively (P < 0.05). © 2014 Japanese Society of Animal Science

6 J. JEONG and I. H. KIM

Table 5 Effect of fermented soybean meal and enzymatically prepared soybean meal on growth performance in weaned pigs

Items

Treatment† FM

Initial BW 6.59 ± 0.26 BW 1 week 8.42 ± 0.49 BW 3 weeks 15.05 ± 1.06a BW 6 weeks 27.35 ± 1.16a Phase I (days 0-7) ADG, g 262 ± 42.6 ADFI, g 298 ± 10.8 G/F 0.84 ± 0.13 Phase II (days 8-21) ADG, g 474 ± 69.2 ADFI, g 647 ± 4.5a G/F 0.73 ± 0.11 Phase III (days 22-42) ADG, g 585 ± 60.9 ADFI, g 871 ± 10.2a G/F 0.67 ± 0.07 Whole period (days 0-42) ADG, g 494 ± 26.3a ADFI, g 701 ± 6.8a G/F 0.71 ± 0.04a

SoELAB

PepSoyGen

Soytide

HP 300

6.59 ± 0.26 8.39 ± 0.48 14.66 ± 0.87ab 27.03 ± 0.81ab

6.59 ± 0.26 8.29 ± 0.74 14.42 ± 0.72b 26.04 ± 1.25c

6.59 ± 0.26 8.40 ± 0.56 14.58 ± 1.22ab 26.53 ± 0.84bc

6.59 ± 0.26 8.40 ± 0.57 15.00 ± 0.84ab 27.35 ± 1.56a

257 ± 55.0 306 ± 15.2 0.84 ± 0.18

243 ± 85.8 303 ± 10.9 0.80 ± 0.28

259 ± 63.7 299 ± 10.9 0.86 ± 0.21

258 ± 68.8 304 ± 18.4 0.85 ± 0.22

448 ± 53.8 634 ± 12.6b 0.71 ± 0.09

438 ± 68.0 633 ± 11.5b 0.69 ± 0.11

441 ± 87.3 627 ± 11.4b 0.70 ± 0.14

472 ± 54.6 631 ± 15.5b 0.75 ± 0.08

589 ± 41.5 857 ± 6.1bc 0.69 ± 0.05

553 ± 77.1 850 ± 16.0d 0.65 ± 0.09

569 ± 64.0 852 ± 8.2cd 0.67 ± 0.07

588 ± 62.7 860 ± 6.2b 0.68 ± 0.07

487 ± 19.0ab 691 ± 5.7b 0.71 ± 0.03a

463 ± 29.5c 686 ± 7.7c 0.68 ± 0.04b

475 ± 21.9bc 685 ± 4.8c 0.69 ± 0.03ab

494 ± 35.0a 691 ± 8.4b 0.72 ± 0.05a

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%; BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; G/F, gain/feed. a,b,c,d Means ± SD in the same row with different superscripts differ (P < 0.05).

Table 6 Effect of fermented soybean meal and enzymatically prepared soybean meal on nutrient digestibility in weaned pigs

Items

1 weeks DM N Energy 3 weeks DM N Energy

Treatment† FM

SoELAB

PepSoyGen

Soytide

HP 300

82.7 ± 1.1ab 80.9 ± 1.3a 82.1 ± 1.5b

82.7 ± 0.9ab 80.3 ± 1.3ab 83.1 ± 1.2ab

81.9 ± 1.2ab 78.4 ± 2.0b 82.2 ± 1.2b

82.9 ± 1.1a 79.7 ± 2.4ab 83.6 ± 1.1a

81.4 ± 1.1b 80.5 ± 1.3ab 83.5 ± 1.1ab

77.1 ± 1.8 75.8 ± 3.1 76.6 ± 1.7

77.1 ± 1.9 74.7 ± 3.5 78.1 ± 3.9

78.8 ± 1.5 74.6 ± 2.4 77.6 ± 2.0

78.6 ± 1.6 74.9 ± 2.6 78.8 ± 2.9

78.6 ± 1.6 75.9 ± 2.7 78.2 ± 2.7

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%; DM, dry matter; N, nitrogen. a,bMeans ± SD in the same row with different superscripts differ (P < 0.05).

Blood profiles and intestinal microflora The effects of the differentially treated SBM on blood profiles are shown in Table 7. No SBM preparations demonstrated any significant differences in WBC and RBC number, lymphocyte %, BUN, creatine or glucose concentrations at weeks 1 and 3 compared with FM treatment, respectively (P > 0.05). In addition, the effects of differentially treated SBM on fecal microflora are shown in Table 8. In general, all treatments led to increased Lactobacillus counts at week 3 compared with FM treatment (P < 0.05); however, no SBM preparation demonstrated any significant differences in E. coli counts at weeks 1 and 3 compared with FM treatment (P > 0.05). © 2014 Japanese Society of Animal Science

Fecal score and excreta noxious gas emission The effect of the differentially treated SBM preparations on fecal score and emission of ammonia, hydrogen sulfide and total mercaptans are shown in Tables 9 and 10, respectively. All of the SBM treatments showed no significant differences in fecal score, except for PepSoyGen on day 5, compared with SoELAB or HP 300 treatment (P < 0.05). None of the SBM treatments showed any significant differences in ammonia or total mercaptans (P > 0.05); however, hydrogen sulfide emission resulting from HP 300 treatment significantly reduced compared with FM treatment (P < 0.05). Animal Science Journal (2014) ••, ••–••

FERMENTED SOYBEAN MEAL IN PIGS

Table 7

7

Effect of fermented soybean meal and enzymatically prepared soybean meal on blood profiles in weaned pigs

Items†

Treatment‡

1 weeks WBC, 103/μL RBC, 106/ μL Lymphocyte, % BUN, mg/dL Creatine, mg/dL Glucose, mg/dL 3 weeks WBC, 103/ μL RBC, 106/ μL Lymphocyte, % BUN, mg/dL Creatine, mg/dL Glucose, mg/dL

FM

SoELAB

PepSoyGen

Soytide

HP 300

24.3 ± 3.9 6.0 ± 0.7 48.0 ± 7.1 12.1 ± 1.8 1.1 ± 0.2 58.0 ± 9.9

25.5 ± 5.4 5.7 ± 0.5 42.7 ± 5.6 11.4 ± 0.8 1.2 ± 0.1 59.0 ± 8.8

25.3 ± 5.1 5.9 ± 0.1 43.1 ± 4.5 13.5 ± 4.2 1.1 ± 0.1 58.5 ± 12.6

25.7 ± 2.8 6.4 ± 1.1 45.9 ± 1.4 12.9 ± 1.1 1.1 ± 0.1 57.8 ± 13.8

25.2 ± 8.0 5.2 ± 1.3 43.1 ± 9.5 13.4 ± 2.3 1.2 ± 0.1 58.3 ± 12.8

14.7 ± 5.4 5.6 ± 0.8 40.6 ± 3.7 12.5 ± 3.1 0.9 ± 0.1 84.3 ± 11.5

12.5 ± 2.4 6.0 ± 0.6 41.1 ± 4.1 11.0 ± 1.5 0.9 ± 0.1 84.5 ± 22.9

13.7 ± 2.7 5.9 ± 1.1 42.1 ± 8.4 11.7 ± 1.6 1.1 ± 0.1 83.5 ± 9.7

13.8 ± 2.9 6.4 ± 0.6 44.6 ± 8.7 11.5 ± 3.3 1.0 ± 0.2 85.3 ± 30.6

13.2 ± 2.8 5.5 ± 0.6 43.7 ± 6.2 13.0 ± 4.0 1.1 ± 0.2 80.3 ± 31.5

†WBC, white blood cell; RBC, red blood cell; BUN, blood urea nitrogen. ‡FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%.

Table 8

Effect of fermented soybean meal and enzymatically prepared soybean meal on fecal microflora in weaned pigs

Items, log10 cfu/g

1 weeks Lactobacillus Escherichia coli 3 weeks Lactobacillus E. coli

Treatment† FM

SoELAB

PepSoyGen

Soytide

HP 300

7.5 ± 0.05 6.4 ± 0.03

7.5 ± 0.19 6.3 ± 0.13

7.6 ± 0.10 6.4 ± 0.05

7.7 ± 0.06 6.3 ± 0.10

7.6 ± 0.08 6.3 ± 0.15

7.4 ± 0.04b 6.4 ± 0.01

7.6 ± 0.06a 6.4 ± 0.04

7.6 ± 0.07ab 6.3 ± 0.10

7.6 ± 0.04a 6.3 ± 0.05

7.6 ± 0.06a 6.4 ± 0.06

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. a,bMeans ± SD in the same row with different superscripts differ (P < 0.05).

Table 9

Effect of fermented soybean meal and enzymatically prepared soybean meal on fecal score in weaned pigs

Item

Fecal score‡ Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Treatment† FM

SoELAB

PepSoyGen

Soytide

HP 300

3.6 ± 0.2 3.4 ± 0.1 3.3 ± 0.2 3.2 ± 0.1 3.1 ± 0.1ab 3.1 ± 0.1 3.1 ± 0.1

3.5 ± 0.1 3.2 ± 0.2 3.3 ± 0.1 3.2 ± 0.1 3.0 ± 0.2b 3.1 ± 0.1 3.0 ± 0.1

3.6 ± 0.2 3.4 ± 0.2 3.2 ± 0.1 3.3 ± 0.0 3.3 ± 0.1a 3.1 ± 0.1 3.0 ± 0.1

3.5 ± 0.2 3.3 ± 0.2 3.4 ± 0.1 3.2 ± 0.1 3.2 ± 0.1ab 3.0 ± 0.1 3.0 ± 0.1

3.4 ± 0.4 3.3 ± 0.3 3.3 ± 0.2 3.2 ± 0.1 3.0 ± 0.1b 3.1 ± 0.1 3.0 ± 0.1

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. ‡Score: 1 hard, dry pellet; 2 firm, formed stool; 3 soft, moist stool that retains shape; 4 soft, unformed stool that assumes shape of container; 5 watery liquid that can be poured. a,bMeans ± SD in the same row with different superscripts differ (P < 0.05).

DISCUSSION This study evaluated the viability of partially replacing FM (up to 50%), which is a prime, highly digestible source of protein with a high content of AA vitamins, and minerals in diets fed to weaned pigs (Jones et al. 2010; Kim et al. 2010), with fermented SBM or enzymatically prepared SBM as alternatives, in an attempt to reduce feed cost while not compromising on growth Animal Science Journal (2014) ••, ••–••

performance. Overall, the results of this study indicate that fermentation of SBM or enzymatic treatment of SBM appears to enrich the SBM for suitable use in diets for weaning pigs. The following improvements of fermented or enzymatically treated SBM over regular SBM are outlined as follows. First, microbial fermentation can reduce the amount of TI and remove glycinin, β-conglycinin, sugars and the majority of oligosaccharides larger than 20 kDa © 2014 Japanese Society of Animal Science

8 J. JEONG and I. H. KIM

Table 10 Effect of fermented soybean meal and enzymatically prepared soybean meal on fecal noxious gas emission in weaned pigs

Items, ppm

Ammonia Hydrogen sulfide Total mercaptans

Treatment† FM

SoELAB

PepSoyGen

Soytide

HP 300

22.8 ± 1.2 8.7 ± 1.4a 8.8 ± 0.6

20.4 ± 3.5 7.8 ± 0.7ab 7.9 ± 0.4

21.1 ± 5.6 7.3 ± 0.6ab 8.5 ± 1.1

20.9 ± 4.9 7.5 ± 0.7ab 8.4 ± 1.3

19.7 ± 4.3 7.1 ± 0.4b 8.4 ± 1.2

†FM, Fish meal (FM) 100%; SoELAB, FM 50%+SoELAB-54 50%; PepSoyGen, FM 50%+ PepSoyGen 50%; Soytide, FM 50%+Soytide 50%; HP 300, FM 50%+HP 300 50%. a,bMeans ± SD in the same row with different superscripts differ (P < 0.05).

(Rojas & Stein 2013), which can reduce the digestibility and utilization of nutrients, thus reducing overall ANF amount (Hong et al. 2004; Min et al. 2004; Yun et al. 2005). Alternatively, removal of oligosaccharide and soluble non-starch polysaccharides (NSP) by ethanol/water extract process results in improvement to intestinal health and growth performance in weaned pigs also (Hancock et al. 1990). In addition, ethanol extraction can achieve a 90% reduction in the oligosaccharides present in SBM, while simultaneously in the process, glycinin and β-conglycinin, the two most important storage proteins in soy, become denatured and lose their antigenic properties (Veldman et al. 1993). Dersjant-Li and Peisker (2008) reported that the most important heat-stable ANFs are proteins exhibiting antigenic effects and oligosaccharides. The differentially treated FSBM used in this study demonstrated lower amounts of raffinose (1/5), stachyose (1/10), and TI compared with commercial SBM. Second, fermentation can reduce the size of peptides in SBM, between 20 and 250 kDa to < 10 kDa by microbial digestion (Hong et al. 2004), resulting in the extensive hydrolysis of protein resulting in more readily available free AA and peptides, thereby improving nutrient availability (Kiers et al. 2000; Cho et al. 2007). SBM composition is generally 48% CP, 35–40% carbohydrates, 7–10% water, 5-6% ash and less than 1% EE (USDA 2009). After microbial fermentation, differentially treated FSBM used in our study demonstrated an increase in CP with a reduction in carbohydrate compared with SBM, which is in agreement with Cervantes-Pahm and Stein (2010) who suggested that FSBM can significantly improve the digestibility of DM and N versus the SBM diet. Moreover, Yang et al. (2007) reported that the analyzed AA composition of FSBM for indispensible AA (IAA) was nearly similar to SBM, but the CP content was higher in FSBM than SBM. In our study, the analyzed total digestible AA (TDAA)/CP % was greater in differentially treated SBM than in FM (> 50.11% vs. 47.12%, respectively) confirming the benefits of fermentation of SBM; however, total IAA/CP % was higher in FM than the differentially treated SBM (47.89% vs. < 45.83%, respectively). Therefore, although fermentation generates more free © 2014 Japanese Society of Animal Science

AA to improve nutrient availability, the amount of essential AA is still lacking in comparison to FM, which means that using FSBM as an alternative for complete FM replacement may not be sufficiently adequate, regarding providing essential AA in the diet perhaps. This point helps to explain the observation that in our study, throughout the whole feeding period (days 0 to 42), all SBM treatments demonstrated reduced ADFI compared with FM treatment. As such, a mixture of FM and FSMB at optimal ratios is required to minimize any growth reduction side effects of using FSMB. Lastly, fermentation can improve nutrient utilization due to manipulation of the gastrointestinal flora, thus potentially affecting digestive and absorptive processes in weaned pigs (Canibe & Jensen 2003). The removal of the low molecular weight carbohydrate fraction through the fermentation and/or extraction process can drastically improve intestinal microbial proliferation resulting in same or higher digestibility of CP and AA in FSBM than FM (Choct et al. 2010; Rojas & Stein 2013). Furthermore, nutritional strategies such as reduction of excess protein and carbohydrate in the diets can contribute to less substrate in the large intestine, reducing the proliferation of pathogens such as E. coli, a major cause of diarrhea (Heo et al. 2009). In our study, FSBM supplementation induced a shift in gastrointestinal microflora, resulting in a significantly increased Lactobacillus count with SoELAB, Soytide and HP 300 compared with FM, respectively. Interestingly though, no difference in ADG and G/F was observed between FM and SoELAB, Soytide and HP 300, during all periods, in this study. The reason for this may be that fermentation increases favorable microbial activity, which may enhance fiber solubility, thereby improving the digestibility of nutrients (Pedersen & Lindberg 2003) compared with SBM.

Conclusion The present study was conducted to determine the comparative efficacy of FM versus fermented SBM (SoELAB, PepSoyGEn, and Soytide) or enzymatically prepared SBM (HP 300) replacing up to 50% of FM, on growth performance, nutrient digestibility and fecal microflora in weaned pigs. Overall, the results from the present study indicate that treated FSBM has a lot Animal Science Journal (2014) ••, ••–••

FERMENTED SOYBEAN MEAL IN PIGS

of potential to serve as a replacement product for FM in diets fed to weaned pigs. However, although fermentation generates more free AA to improve nutrient availability, the amount of essential AA is still lacking in comparison to FM, which means that using FSBM as a complete alternative to FM may not be sufficiently adequate, regarding providing essential AA in the diet perhaps. As such, a FSBM and FM mixture in the diet may be ideal which would reduce the amount of FM commonly used in diets fed to weaned pigs. Future research into feeding studies of weaned pigs with diets involving various FSBM and FM mixture rations are needed to determine the optimal ratio and confirm the potential of FM replacement with FSBM.

ACKNOWLEDGMENTS The authors wish to thank Peter Lee for his English correction and help with manuscript preparation.

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