Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae mixed fermented feed on the enhanced growth performance of broilers

METABOLISM AND NUTRITION Effects of Bacillus subtilis var. natto and Saccharomyces cerevisiae mixed fermented feed on the enhanced growth performance of broilers K.-L. Chen,* W.-L. Kho,* S.-H. You,* R.-H. Yeh,* S.-W. Tang,* and C.-W. Hsieh†1 *Department of Animal Science, National Chiayi University, Taiwan; and †Department of Microbiology and Immunology, National Chiayi University, Taiwan FBac+Sac, Bac powder (PBac), Sac powder (PSac), and Bac+Sac powder (PBac+Sac). The results from trial 1 showed that FBac+Sac increased BW and feed intake (P < 0.05) in 21- and 39-d-old chickens. The water-added group showed decreased BW, weight gain, and feed intake (P < 0.05). Trial 2 showed that FBac+ Sac increased gross energy availability (P < 0.05). Trial 3 showed that FBac+Sac increased 21- and 39-d-old BW and weight gain (P < 0.05). Diets supplemented with probiotic powder or fermented with Sac did not improve broiler growth performance (P > 0.05). The growth performance improvement of the FBac+Sac treatment was probably not due to the added water, probiotic powder inclusion, or through single-strain fermentation, but due to the 2-stage fermentation process using Bac and Sac strains.

Key words: Bacillus subtilis var. natto, Saccharomyces cerevisiae, fermented feed, broiler 2009 Poultry Science 88:309–315 doi:10.3382/ps.2008-00224

INTRODUCTION

lipase, can improve growth performance (Santoso et al., 2001). Saccharomyces with protein digestion and high acidic capacity could prevent antimicrobial-associated diarrhea (Surawicz et al., 1989; Bleichner et al., 1997). Diets supplemented with 0.5% dried B. subtilis fermented product or probiotic powder improved weight gain and feed efficiency (Santoso et al., 1995, 2001; Jin et al., 1996). Diets supplemented with yeast (Saccharomyces cerevisiae) cell-wall components can improve growth performance in chickens (Karaoglu and Durdag, 2005; Zhang et al., 2005). Utilizing probiotics to preserve feedstuffs or feed fermentation such as liquid fermenting feed or silage for ruminants and pigs have been applied for many years (Cumby, 1986; Boguhn et al., 2006), with beneficial effects (Besong et al., 1996; Yang and Beauchemin, 2006). However, there is limited information on the use of fermented feeds in chickens. Therefore, this study investigated the beneficial effects of combined 2-stage fermentation feed inoculated with high proteolytic capacity Bacillus subtilis var. natto N21 (Bac) in the first stage, and high acidic capacity Saccharomyces cerevisi-

Antibiotics have been used as feed additives to improve growth performance and control disease in animals. However, antibiotic use tends to produce antibiotic resistance and residues in animal products. Since 2006, antibiotics have been banned for use as feed additives in the European Union. Probiotics have therefore become important as replacement feed additives (Steiner, 2006). The major probiotic strains include Lactobacillus, Saccharomyces, Bacillus, Streptococcus, and Aspergillus (Tannock, 2001). Presently Bacillus (Jin et al., 1996), Lactobacillus (Jin et al., 1996; Yeo and Kim, 1997), and Saccharomyces (Zhang et al., 2005) are the major strains applied in broilers. Diets supplemented with Bacillus subtilis, which secrete protease, amylase, and ©2009 Poultry Science Association Inc. Received June 3, 2008. Accepted October 14, 2008. 1 Corresponding author: [email protected]

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ABSTRACT Bacillus subtilis var. natto N21 (Bac; for greater proteolytic capacity) and Saccharomyces cerevisiae Y10 (Sac; for greater acidic capacity) were applied to produce a 2-stage combined fermentation feed. This study investigated whether the enhancement of Bac+Sac fermented feed on broiler growth performance was due to the probiotics per se or due to the fermentation process. Trial 1 included 1-d-old broiler chicks (n =144) randomly assigned to control, water added (same as in the fermentation feed, 23%), and Bac+Sac fermented feed (FBac+Sac) treatments with 4 replicates. Trial 2 included 21-d-old broiler chickens (n = 12) assigned into control and FBac+Sac groups for a metabolic trial for nutrient availability. Trial 3 included 1-d-old male broiler chicks (n = 216) randomly assigned into 6 treatments with 3 replicates. Treatments included a control, Sac fermented feed (FSac),

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ae Y10 (Sac) in the second-stage fermentation. Broiler feeding trials were conducted to investigate if the beneficial effects of Bac+Sac fermented feed were due to the probiotics or to the fermentation process.

MATERIALS AND METHODS Fermented Feed Preparation

Trial 1 Bird Management and Experimental Design. One hundred forty-four 1-d-old Arbor Acres broiler chicks were randomly assigned into control, water added (same as in the fermented feed, 23%), and Bac+Sac fermented feed (FBac+Sac) with 4 replicates. Chicks were raised in a battery brooder from 0 to 21 d and in floor pens (3.0 m × 1.7 m) from 21 to 39 d. Feed (Table 1) and water were provided ad libitum. Bird management followed the Arbor Acres broiler management manual (Arbor Acres, 2001).

Measurements and Analysis Growth Performance. Chicken BW and feed intake were recorded each week to calculate weight gain and feed conversion. Carcass Traits. Twelve chicks each from the control and FBac+Sac groups were euthanized at 21 and 39 d of age to measure the weights of the liver, proventriculus, and gizzard, abdominal fat (from the gizzard to celiac fat), breast muscle, thigh muscle (deboned thigh drumstick muscle), spleen, thymus, and bursa. Intestinal Histology. Two-centimeter fragments of the duodenum (posterior to gizzard), jejunum (anterior to Meckel’s diverticulum), and ileum (anterior to ileocecal junction) were collected after 21 d on the feeding trial. The intestinal fragments were prepared and examined using an optical microscope (Labophot-2, Nikon, Tokyo, Japan) according to Chiou et al. (1999). The villus height, villus perimeter, crypt depth, and muscle layers of the small intestine were measured using the method of Uni et al. (1995) with 10 points for each measurement. Villus height was based on the top of the crypt using the lamina propria of the villus. The villus perimeter was based on the villus circumference and the distance between neighboring villi. Crypt

Item Ingredient, % Yellow corn, grain Soybean meal, 44% Fish meal, 65% Soybean oil Dicalcium phosphate Limestone, pulverized Salts dl-Methionine Vitamin premix1 Mineral premix2 Total Calculated value CP, % ME, kcal/kg Ca, % Available P, %

d 0 to 21 49.21 39.9 2.0 6.15 0.51 1.63 0.3 0.1 0.1 0.1 100 23 3,100 0.90 0.45

d 21 to 39 56.0 35.4 0 5.0 2.0 1.0 0.3 0.1 0.1 0.1 100 20 3,200 0.85 0.42

1 Vitamin premix supplied per kilogram of diet: vitamin A, 3,000 IU; vitamin D3, 400 IU; vitamin E, 10 IU; vitamin K3, 1 mg; vitamin B1, 3.6 mg; vitamin B2, 5.4 mg; vitamin B6, 7.0 mg; Ca-pantothenate, 20.0 mg; niacin, 70 mg; biotin, 0.3 mg; folic acid, 1.1 mg; vitamin B12, 0.02 mg. 2 Mineral premix supplied per kilogram of diet: Cu (CuSO4·5H2O, 25.45% Cu), 8 mg; Fe (FeSO4·7H2O, 20.09% Fe), 80 mg; Mn (MnSO4·H2O, 32.49% Mn), 60 mg; Zn (ZnO, 80.35% Zn), 40 mg; Se (NaSeO3, 45.56% Se), 0.15 mg.

depth was the shortest vertical distance from the villus contact point to the mucous membrane. The muscle layer was the shortest vertical distance from the point between the epimysium and annularity muscle to the mucous membrane. pH of Digestive Tract Contents. The pH of the contents of the crop, proventriculus, duodenum (between the gizzard and end of the duodenal loop), jejunum (15 cm anterior to Meckel’s diverticulum), ileum (between Meckel’s diverticulum and ileo-cecal junction), and cecum were measured using a portable pH meter (digital pH meter, Goodly, Taiwan). Intestinal Tract Microflora Population. The duodenum and cecum contents were diluted 10-fold with buffered peptone water and vortexed for 2 min; the supernatant was preserved at −80°C. One hundred microliters of supernatant was smeared onto MacConkey agar and Lactobacilli MRS agar and incubated at 37°C for 24 h or 37°C with 13% CO2 for 48 h, respectively. Intestinal Enzyme Activity. The intestinal contents were collected after the feeding trial. The samples were diluted 10-fold (based on sample weight) with 0.9% NaCl and homogenized. The samples were then centrifuged at 2,000 × g for 30 min. The resulting supernatant was a crude enzyme solution. α-Amylase (EC 3.2.1.1) and lipase (EC 3.1.1.3) were measured using kinetic methods as recommended by the German Society for Clinical Chemistry (1972) by using an automatic blood chemical analyzer (Cobas Mira Plus, Roche, Basel, Switzerland) with Roche testing kits. Neutral protease activity was analyzed using a modified Boyce method (Boyce, 1986). The crude enzyme fluid protein content was analyzed using a modified Bradford method (Bradford, 1976).

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Feed was supplemented with Bac powder (106 cfu/g of feed) and 10% water at 25 to 35°C for a 2-d aerobic fermentation in the first stage and then with Sac (106 cfu/g feed) and 13% water at 25 to 35°C for a 3-d anaerobic fermentation in the second stage (Kho, 2006). After fermentation, lactic acid and acetic acid were the major acids produced and the pH value declined 15% (control, pH 6.5; Bac+Sac fermented feed, pH 5.5), and the amounts of Bac and Sac were 2.2 × 106 and 2.3 × 108 cfu/g of feed, respectively.

Table 1. Composition of the basal diet

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Table 2. Effects of FBac+Sac on growth performance of broilers at d 0, 21, and 39 (trial 1) Item BW, g/bird d0 d 21 d 39 Weight gain, g/bird d 0 to 21 d 21 to 39 d 0 to 39 Feed intake,2 g/bird d 0 to 21 d 21 to 39 d 0 to 39 Feed conversion, feed intake/weight gain d 0 to 21 d 21 to 39 d 0 to 39

Control

Water

FBac+Sac

SEM

37.5 713b 1,793b

38.2 701b 1,642c

38.2 756a 1,943a

0.21 1.23 2.60

675b 1,079b 1,756b

663b 949c 1,604c

718a 1,184a 1,905a

1.23 2.30 2.60

973 1,971b 2,944b

933 1,721c 2,654c

961 2,136a 3,097a

3.42 3.87 4.45

1.44 1.83 1.68

1.41 1.82 1.65

1.34 1.80 1.63

0.13 0.15 0.10

Means in the same row with different superscripts are significantly different (P < 0.05). FBac+Sac: supplementation with Bacillus subtilis N21powder (106 cfu/g of feed) and added 10% water for a 2-d fermentation, and then supplementation with Saccharomyces cerevisiae Y10 powder (106 cfu/g of feed) and added 13% water for 3-d fermentation. 2 Feed intake in both the water and the FBac+Sac groups contained 34% moisture, and was converted from an as-fed basis to an air-dry basis with 11% moisture as control. 1

Trial 2 Metabolic Trial for Nutrient Availability. Twelve 21-d-old broiler chicks with similar BW were selected from the control and FBac+Sac groups from trial 1. These birds were housed in individual cages (30 × 25 × 40 cm) for the nutrient availability trial. Excreta were collected for 4 d after a 3-d adaptation period. Water and feed were provided ad libitum in the adaptation period. During the collection period, feed was provided at 70% of the amount consumed in the adaptation period; Cr2O3 was added to the experimental diet as an indigestible indicator. The calculation formula was as follows: digestibility (%) = 100 − 100 × [indicator in feed (%)/indicator in excreta (%)] × [nutrient concentration in excreta (%)/nutrient concentration in feed (%)]. Moisture (oven-drying method, method 930.15) and CP (method 990.03) analyses were performed according to AOAC (1990) methods, and gross energy was measured with an adiabatic bomb calorimeter (model 356, Parr Instrument Company, Moline, IL). Analysis of Cr2O3 was performed using atomic absorption spectrophotometry (model 2380, Perkin-Elmer, Wellesley, MA) as described by Williams et al. (1962), and the digestibility of each nutrient was measured.

Trial 3 Bird Management and Experimental Design. Two hundred sixteen 1-d-old male broiler chicks were randomly to the following treatments: 1) control (same as in trial 1); 2) Sac fermented (diet supplemented with 106 cfu of Sac/g of feed, 23% water was added by weight, and fermentation occurred for 3 d, FSac); 3) FBac+Sac (same as in trial 1); 4) Bac powder (diet supplemented with 106 cfu of Bac/g of feed, PBac); 5)

Sac powder (diet supplemented with 108 cfu of Sac/g of feed, PSac); 6) Bac+Sac powder (diet supplemented 106 cfu of Bac/g of feed and 108 cfu of Sac/g of feed, PBac+Sac)

Measurements and Analysis Growth Performance. The measurements and analyses were identical to those of trial 1.

Statistical Analysis Variances among the treatments were calculated using the GLM procedure (SAS Institute, 1990). Duncan’s new multiple-range test was used to compare the means according to Steel and Torrie (1980).

RESULTS AND DISCUSSION Trial 1 Growth Performance. Table 2 presents the FBac+Sac effect on broiler growth performance. Feeding FBac+Sac increased BW and weight gain of 21- and 39-d-old chickens and feed intake (P < 0.05) from 21 to 39 d and 0 to 39 d. However, the water-supplemented group had decreased BW, weight gain, and feed intake from 21 to 39 d and from 0 to 39 d (P < 0.05). Santoso et al. (1995, 2001) reported that diets supplemented with 0.5% B. subtilis fermentation product could improve broiler the growth performance. Zhang et al. (2005) reported that diets supplemented with 0.3% yeast (S. cerevisiae) cell components improved broiler BW gain, feed intake, and feed efficiency. In this study, fermented feed increased (P < 0.05) BW and weight gain at 21 and 39 d, with FBac+Sac increasing broiler

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a–c

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Table 3. Effects of FBac+Sac on carcass traits in broilers at d 21 and d 39 (trial 1) Relative organ weight, g/100 g of BW

FBac+Sac

SEM

0.94 1.64

1.25 1.49

0.18 0.19

2.31a 1.71

2.06b 1.66

0.13 0.12

4.27 3.86

4.16 3.86

0.65 0.15

2.13b

2.38a

0.08

9.78

9.89

0.20

7.85

7.75

0.23

0.08 0.11

0.07 0.11

0.04 0.04

0.45 0.53

0.54 0.57

0.07 0.08

0.25 0.19

0.22 0.20

0.09 0.08

a,b Means in the same row with different superscripts are significantly different (P < 0.05). 1 FBac+Sac = supplementation with Bacillus subtilis N21powder (106 cfu/g of feed) and added 10% water for a 2-d fermentation, and then supplementation with Saccharomyces cerevisiae Y10 powder (106 cfu/g of feed) and added 13% water for 3-d fermentation.

BW by 6.3 and 8.3% at 21 and 39 d, respectively. These results agreed with the results described above (Santos et al., 1995, 2001; Zhang et al., 2005). The increase in fermented feed intake during d 0 to 39 could be attributed to the palatability of the fermented feed with its special aroma and acidification. Kho (2006) indicated that the pH value of FBac+Sac decreased 15% (control, pH: 6.5, FBac+Sac, pH: 5.5), and the major acidic contents produced were lactic acid (control: 4.13 mg/mL vs. FBac+Sac: 16.48 mg/g) and acetic acid (control: 0 mg/g vs. FBac+Sac: 3.71 mg/g). Diets supplemented with acidifier (3 g/kg) increased feed intake in broilers, and thus increased BW (Lückstädt et al., 2004). The increased weight gain in the FBac+Sac group in this study was attributed to the palatability of the fermented feed; hence, the feed intake increased from d 21 to 39 and d 0 to 39. The fermented feed was inoculated with probiotics with 23% added water during fermentation. This trial investigated whether the enhanced broiler growth performance was due to the Bac+Sac fermented feed or to the 23% added water. The 23% added water did not affect growth performance from 0 to 21 d (P > 0.05). However, feed intake at d 21 to 39 and d 0 to 39 did decrease (P < 0.05) and thus decreased BW and weight gain (P > 0.05). This may because inclusion of water only could not acidify the diet to preserve the feed, resulting in a putrefied feed that decreased growth performance. Hence, it was clear that the increase in

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Abdominal fat d 21 d 39 Liver d 21 d 39 Intestines d 21 d 39 Proventriculus and gizzard d 39 Left thigh d 39 Left breast d 39 Spleen d 21 d 39 Thymus d 21 d 39 Bursa d 21 d 39

Control

growth performance was due to Bac+Sac fermentation, not to the 23% added water. Carcass Traits. Table 3 presents the effects of FBac+Sac on broiler carcass traits. The results showed that Bac+Sac fermented feed decreased relative liver weight at d 21 (P < 0.05), and increased the relative weights of proventriculus and gizzard at d 39 (P < 0.05). Santoso et al. (1995) reported that dietary supplemented B. subtilis fermented product at 10 or 20 g/ kg of feed could increase relative abdominal fat weight, but their results did not agree with those obtained in this study. The FBac+Sac treatment increased relative proventriculus and gizzard weights (P < 0.05) because of increased feed intake 8.3% and 5.2%, respectively, from d 21 to 39 and from d 0 to 39. The FBac+Sac treatment decreased relative liver weight at d 21 (P < 0.05), but had no influence at d 39 (P > 0.05). However, feed intake significantly increased during d 21 to 39 (P < 0.05), but had no influence from d 0 to 21 (P > 0.05). The liver is the main lipogenic organ in poultry. Whether this result implied that the FBac+Sac will decrease the relative weight of the liver needs further research. pH Value, Lactobacillus-Like, and Coliform Population of Digestive Tract Contents. The pH values of crop, proventriculus, duodemun, jejunum, and ileum did not exhibit significant differences between all treatments (P > 0.05). The lactobacillus-like and coliform populations in the duodenum and cecum were not significantly different among treatments (P > 0.05); thus, these results were not shown. Diets fermented with Bac+Sac could reduce pH. However, there was no effect on pH values in the digestive tract. This may be because 1) the main fermented feed acids were acetic acid and lactic acid but they were easily volatilized or absorbed by the gastrointestinal tract; 2) feed was withdrawn from the chickens for 12 h before slaughter so that no digestive tract contents would be present. Hence, Bac+Sac fermented feed had no influence on the digestive tract pH values. Probiotics suppress the survivability of pathogenic microflora such as Escherichia coli and Salmonella at pH 4.0 (Giannella et al., 1972; Jin et al., 1996, 1998). However, in this study, fermented feed could not reduce pH. Hence, the coliform population was not suppressed. Enzyme Activity and Histology of Intestinal Tract. The FBac+Sac treatment had no influence on enzyme activity, villus height, villus perimeter, crypt depth, and muscle layers of duodenum, jejunum, and ileum during the experiment period (P > 0.05). Thus, these data are not shown. Bacillus subtilis and S. cerevisiae can secrete protease, amylase, and lipase (Santoso et al., 2001; Koh et al., 2002). However, there was no effect on protease, amylase, and lipase activity in this study. This was probably because of the 12-h feed and water withdrawal before slaughter.

PROBIOTIC FERMENTATION FEED AND GROWTH PERFORMANCE OF BROILERS 1

Table 4. Effects of FBac+Sac on nutrient availability in broilers (trial 2) Item, % DM CP Gross energy

Control

FBac+Sac

SEM

83.1 66.1 84.1b

83.2 66.4 85.3a

0.45 0.41 0.38

a,b Means in the same row with different superscripts are significantly different (P < 0.05). 1 FBac+Sac = supplementation with Bacillus subtilis N21powder (106 cfu/g of feed) and added 10% water for a 2-d fermentation, and then supplementation with Saccharomyces cerevisiae Y10 powder (106 cfu/g of feed) and added 13% water for 3-d fermentation.

Trial 2 Metabolic Trial for Nutrient Availability. The FBac+Sac effect on nutrient availability in broilers is shown in Table 4. Fermented feed increased gross energy availability (P < 0.05). Bacillus subtilis secrete protease, amylase, and lipase (Santoso et al., 2001) and can thus be beneficial to

digestion. The FBac+Sac treatment did increase gross energy availability (P < 0.05) compared with the control group. In agreement with results from trial 1 (Table 2), the fermented feed group increased weight gain with no increase in feed intake from d 0 to 21.

Trial 3 Growth Performance. The effect of FBac+Sac or probiotics powder on growth performance in broilers is shown in Table 5. The FBac+Sac treatment increased BW and weight gain at d 21 and 39 (P < 0.05). The FSac and probiotic powder groups showed no difference compared with the control group (P > 0.05). The FBac+Sac treatment increased feed conversion compared with FSac and PBac+Sac (P < 0.05). The FBac+Sac treatment increased BW in 21- and 39-d-old chicks by 5.4 and 15.4%, respectively. These results agreed with the results from trial 1 and again proved that FBac+Sac can improve growth performance in broilers. The FSac treatment did not improve growth performance (P > 0.05) or feed conversion; both were lower than that for the control group (P < 0.05). Therefore, FBac+Sac must be made using 2-stage fermentation using B. subtilis var. natto Bac in the first stage and S. cerevisae Y10 in the second stage to obtain the increased growth performance in broilers. The feed proteins were degraded into small molecules by protease from B. subtilis (Tsai and Hsieh, 2005). Therefore, single-strain fermentation by S. cerevisiae could not improve broiler growth performance without fermentation by B. subtilis var. natto.

Table 5. The effects of fermented feed or probiotic powder on growth performance in broilers (trial 3) Fermented feed1 Item BW, g/bird d0 d 21 d 39 Weight gain, g/bird d 0 to 21 d 21 to 39 d 0 to 39 Feed intake,3 g/bird d 0 to 21 d 21 to 39 d 0 to 39 Feed conversion, feed intake/weight gain d 0 to 21 d 21 to 39 d 0 to 39 a –c

Control

FSac

FBac+Sac

Probiotic powder2 PBac

PSac

PBac+Sac

SEM

40.6 722b 1,801b

41.1 706bc 1,864b

40.4 761a 2,090a

41.2 715bc 1,836b

40.8 695c 1,844b

41.2 728b 1,857b

0.30 1.25 2.25

681bc 1,078b 1,760b

664bc 1,152b 1,822b

721a 1,328a 2,050a

673bc 1,123b 1,795b

654c 1,149b 1,803b

687b 1,129b 1,816b

1.24 2.08 2.25

960 2,002 2,962

996 2,113 3,109

1006 2,186 3,191

955 2,037 2,992

935 1,955 2,891

1007 2,134 3,138

4.24 7.53 7.70

1.41ab 1.86 1.68ab

1.50a 1.84 1.71a

1.40b 1.65 1.56b†

1.42ab 1.81 1.66ab

1.43ab 1.70 1.60ab

1.46ab 1.90 1.73a

0.13 0.22 0.16

Mean in the same row with different superscripts are significantly different (P < 0.05). FSac = supplementation with Saccharomyces cerevisiae Y10 (Sac) powder (106 cfu/g of feed) and added 23% water for 3-d fermentation; FBac+Sac = supplementation with Bacillus subtilis N21 (Bac) powder (106 cfu/g of feed) and added 10% water for 2-d fermentation, and then supplementation with Sac powder (106 cfu/g of feed) and added 13% water for 3-d fermentation. 2 PBac = supplementation with Bac powder (106 cfu/g of feed); PSac = supplementation with Sac powder (108 cfu/g of feed); PBac+Sac = supplementation with Bac powder (106 cfu/g of feed) and Sac powder (108 cfu/g of feed). 3 Feed intake in the both the water and the FBac+Sac groups both contained 34% moisture, and was converted from an as-fed basis to an air-dry basis with 11% moisture as control. †FBac+Sac vs. control (P < 0.10). 1

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Crypt depth is highly relevant to epithelium activity (Noy and Sklan, 1997), which also signifies the strength of the intestinal hyperplasia and relevant decrease of enzyme activity in the villus brush border (Hampson, 1986).. The fermented feed had no effect on villus height, villus perimeter, and crypt depth in this study. The increase in muscle layer showed intestinal peristalsis enhancement. The Bac+Sac fermented feed group had greater feed intake but there was no effect on muscle layer thickness.

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ACKNOWLEDGMENTS The authors thank the Council of Agriculture (Taipei, Taiwan) for financially supporting this project (project no. 95AS-5.1.3-AD-U1).

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Bacillus subtilis var. natto is an aerobic microorganism. Feed fermented using Bac with a high water content will result in putrefaction during long-term preservation. Therefore, Bac only was not considered to produce the fermentation feed. To prove that the efficacy of FBac+Sac was because of the amount of probiotics or not, feed was supplemented with probiotic powder based on B. subtilis at about 106 cfu/g and S. cerevisiae at about 108 cfu/g in the Bac+Sac fermented feed in this trial. However, PBac, PSac, and PBac+Sac probiotic powder had no effect on growth performance (P > 0.05). Jin et al. (1996) reported that diets supplemented with 0.1% B. subtilis powder could increase weight gain in 10-d-old male Arbor Acres broiler chicks in a high temperature and humidity environment. Zhang et al. (2005) reported that diets supplemented with S. cerevisiae (1.3 × 1010 cfu/g) could improve weight gain and feed efficiency in Ross broilers. Mutus et al. (2006) reported that diets supplemented with Bacillus licheniformis and B. subtilis could improve avian × avian chick feed efficiency. These results did not agree with those reported in the current trial. This may be attributed to the environment, strains, and probiotic microflora population. As a result, diets supplemented with probiotic powder with the same microflora population as FBac+Sac could not improve growth performance (P > 0.05). This may be because 1) fermented feed could degrade large molecule material; 2) microflora may produce substances that are beneficial to broiler growth and health; or 3) the probiotics in fermented feed were activated, whereas the probiotic powders were not. Probiotic powder is unfavorable for retention in the intestines because of the shorter gastrointestinal tract length in broilers.

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