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Probiotic (Bacillus coagulans) cells in the diet benefit the white shrimp Litopenaeus vannamei.

ABSTRACT We evaluated the effects of the dead probiotic Bacillus coagulans as a diet additive on growth performance, survival rate, digestive enzymes, and meat quality of the white shrimp Litopenaeus vannamei. Two treatment groups (T-1 and T-2) and a control group, each with 3 replicates, were established. The shrimp in the control were fed a basal diet, and those in T-1 and T-2 were fed with a basal diet containing viable and dead probiotic cells, respectively. After 50 days of culture, the addition of the probiotic resulted in greater (P < 0.05) final weight and daily weight gain, and survival rate. As for growth performance, the highest values (P < 0.05) were observed in T-1. Higher activities (P < 0.05) of protease, amylase, and lipase were also found in T-1 compared with T-2 and the control. No significant differences appeared in the muscle compositions of moisture, crude protein, and ash. However, the group that received viable probiotic showed greater (P < 0.05) muscle crude fat composition than that seen in the control group. In addition, greater (P < 0.05) inosinic acid content was observed in T-1. Our results showed that the dietary supplementation of both viable and dead probiotic, especially viable, can improve growth and survival rates of white shrimp.

KEY WORDS: probiotic, Penaeus vannamei, shrimp, growth performance, digestive enzyme


With the increasing demand for environmentally friendly aquaculture, the use of probiotics such as Bacillus and yeast in aquaculture is widely accepted (Wang et al. 2008). A number of probiotics are available commercially and have been introduced to fish, shrimp, and molluscan farming as feed additives, or are incorporated into pond water (Noh et al. 1994, Moriarty 1998, Rengpipat et al. 1998, Scholz et al. 1999, Alavandi et al. 2004, Gullian et al. 2004, Wang et al. 2005, Planas et al. 2006, Wang & Xu 2006, Tseng et al. 2009, Wang & Gu 2010). Probiotics, by the generally accepted definition, are a live microbial adjunct that has a beneficial effect on the host by competitive exclusion of pathogenic bacteria, by modification of the host-associated or ambient microbial community, by improved use of the feed or enzymatic contribution to digestion, or by enhancement of the immune response against pathogenic microorganisms (Verschuere et al. 2000, Balcazar et al. 2006). Therefore, high levels of viable microorganisms are recommended in probiotics for efficacy, although specific numbers are not mentioned in the definition (Gatesoupe 1999).

However, according to Irianto and Austin (2002), a probiotic is an entire microorganism or components of a microorganism that is beneficial to the health of the host. This definition is a lengthy way of describing a probiotic apart from the requirement of the probiotic to be a live culture, and thus it would allow for certain suggested immunostimulants (Smith et al. 2003). A previous study indicates further that the protective effect of probiotics is at least mediated in part by the probiotics' own DNA, rather than their metabolites or their ability to colonize the digestive tract (Rachmilewitz et al. 2004). This suggests that the benefits of probiotics not only come from viable probiotics but also from nonviable, "dead" probiotics. Irianto and Austin (2003) also observed the potential effect of dead probiotic cells for control of furunculosis in rainbow trout, Oncorhynchus mykiss. Few studies have been carried out on the use of dead probiotic Bacillus coagulans cells as diet additives in the Pacific white shrimp Litopenaeus vannamei. Therefore, the principal objective of this study was to examine the growth performance, survival rate, digestive enzymes, and meat quality of the white shrimp in response to the viable and dead probiotic B. coagulans supplemented in the diet.


Probiotic Strain and Diet

The probiotic B. coagulans was prepared in the laboratory according to Wang (2009), and examined routinely for purity based on morphological and biochemical characteristics (Dong & Cai 2001). The strains on normal nutrient agar (Difco, Shanghai, China) by spore staining with the spread plate technique were cultured and counted (Marshall & Beers 1967). For probiotic treatment, bacteria were harvested and prepared after their suspension in sterile phosphate-buffered saline (PBS) solution (pH 7.2; Sangon, China). The dead probiotic cells obtained after heating the microorganisms at 100[degrees]C for 30 min were incubated in medium, as described, in a shaking incubator at 28[degrees]C to confirm the absence of bacterial growth. The ingredients and chemical composition of the basal diets used in the experiment were described by Deshimaru and Kuroki (1974) and Wang et al. (2002), and the basal diet formulation is given in Table 1. The dietary ingredients were mixed with the same cell number of live or dead probiotics, pelletized, air-dried by an electric fan at room temperature, and kept at -20[degrees]C until used. The shrimp in the control group were fed the basal diet during the entire trial period. The shrimp in treatment 1 (T-1) and treatment 2 (T-2) were fed with diets containing live and dead probiotic cells with a final concentration of [10.sup.7] colony forming units (cfu)/g feed, respectively.

Experimental Design

Healthy white shrimp (L. vannamei) were obtained from the Shrimp Hatchery of Xiaoshan, Hangzhou, China; maintained in a concrete tank (3 x 2 x 1 m); and fed the basal diet 3 times daily for 2 wk. Shrimp with similar size (0.86 [+ or -] 0.02 g) were stocked randomly in 9 tanks at an initial density of 50 shrimp per tank (500 L water), with 3 replicate tanks for each of the 2 treatments (T-1 and T-2) and the control. Each tank was supplied with running seawater that had been cotton filtered, with a flow rate of 1 L/min, which was then passed successively through a tungsten heater and a degassing column packed with plastic rings. The temperature was maintained at 25 [+ or -] 1 [degrees]C and salinity at 32-33[per thousand]. A photoperiod of 12 h light and 12 h darkness was maintained during the entire trial. The dissolved oxygen level in the tanks was maintained above 6 mg/L by an air pump (ADP-2200, Jinlai Pump Factory, China), and was measured using a Hach kit (model DREL 2400; Hach Company, CO). The shrimp were fed 2 times daily at 0800 Ha and 1700 HR with a commercial diet. The daily feeding rate was about 3% of the total body weight (in grams) and was adjusted according to actual food intake of the white shrimp. Every day the remaining diet of each tank was collected by siphoning before the second daily feeding. Every third day, each tank was cleaned of feces, and the water was partially changed (about 50%). Furthermore, the mortality rate in each tank was recorded daily. At the end of the 50-day experiment, percent shrimp survival was determined.

Sampling and Analytical Methods

At the end of the 50-day experiment, the shrimp were starved for 24 h before processing. Wet weight of all shrimp was measured using an electronic balance (Shanghai Spectrum Co., China). The daily weight gain (measured in grams per day) was calculated as (Final weight Initial weight)/50. The relative weight gain rate was calculated using the following formula: (Final weight initial weight)/Initial weight.

Ten white shrimp from each treatment or control were collected randomly and anesthetized for the digestive enzyme assay. Shrimp dissection was carried out on ice, and the intestinal contents were homogenized in PBS solution (pH 7.5; Sangon, China), 1 g/10 mL, using a handheld glass homogenizer. Last, the homogenate was centrifuged at 4[degrees]C at 15,000g for 15 min and the supernatant was stored at -70[degrees]C (Forma 702; Thermo) until enzyme assays were done. All enzymatic assays were conducted within 48 h after extraction. Protease activity was evaluated according to Lowry et al. (1951) using Folinphenol reagent, and amylase activity was measured according to Jiang (1982) and Worthington (1993) using iodine solution to reveal nonhydrolyzed starch. Lipase activity was determined based on measurement of fatty acid release resulting from enzymatic hydrolysis of triglycerides in a stabilized emulsion of olive oil (Borlongan 1990, Jin 1995). Enzyme activity was measured as the change in absorbance using a Shimadzu 160-UV spectrophotometer, and was defined as enzyme units per gram of intestine content.

The proximate composition, including crude protein, crude fat, ash (muffle furnace, oven incineration at 550[degrees]C for 24 h), and moisture (oven drying at 105[degrees]C for 24 h), of the basal diet and shrimp muscle was determined using the standard methodology of the Association of Official Analytical Chemists (1990). Crude protein was determined using the Kjeltec Analyzer Unit (2300; Sweden) after acid digestion and crude fat were measured using the Soxtec Auto Extraction Unit (2050; Sweden). Gross energy of the basal diet was determined with an adiabatic bomb calorimeter (PARR 1281).

The contents of inosinic acid (inosine 5'-monophosphate (IMP)) in the shrimp muscle were determined using high-performance liquid chromatography (HPLC; Waters 515) with the modification method of Wu et al. (2005). Six shrimp were collected randomly from each group for the IMP assay. One gram of muscle from each shrimp was homogenized with 5 mL 0.6 M perchloric acid using a handheld glass homogenizer at 0[degrees]C. The homogenate was centrifuged at 3,000g for 10 min, and 10 mL of the supernatant was neutralized immediately to pH 6.5 with 1 M potassium hydroxide. After standing at 0[degrees]C for 30 min, the solution was filtered through a 0.22-[micro]m Millipore filter before being analyzed by HPLC.

Differences among the means were tested for statistical significance using 1-way analysis of variance (ANOVA) followed by Duncan's multiple range test. A significance level of P < 0.05 was used in the current study.


Growth Performance and Survival Rate

There was no significant difference (P > 0.05) in initial weight of the white shrimp among the treatment and control groups (Table 2). However, the administration of the live probiotics for 50 days resulted in significantly higher (P < 0.05) final weight and daily weight gain in shrimp than in those from the dead probiotic group, which in turn was significantly (P < 0.05) higher than in those from the control group (Table 2). As for the survival rate, the probiotic treatment groups also showed a significantly higher value (P < 0.05) than that of the control group (Table 2). However, there was no significant difference in survival rate between T-1 and T-2 (87.11 [+ or -] 3.42% and 82.22 [+ or -] 1.39%, respectively; Table 2).

Digestive Enzymes

Specific enzyme activities for protease, amylase, and lipase of shrimp treated with or without probiotic as a diet additive are shown in Figures 1, 2, and 3. In the shrimp fed with live probiotic cells (T-1), significantly higher activities (P < 0.05) of these 3 enzymes were found compared with those fed with dead probiotic cells (T-2) and without probiotic cells (control). However, no significant differences (P > 0.05) were found in the activities of protease, amylase, and lipase for T-2 shrimp compared with control shrimp.

Meat Quality

The group that received viable probiotic cells had a significantly higher (P < 0.05) crude fat composition in the muscle than that of the control group (Table 3). However, there was no significant difference (P > 0.05) in crude fat composition between T-1 and T-2 shrimp (1.94 [+ or -] 0.12% and 2.15 [+ or -] 0.12%, respectively). In addition, no significant difference (P < 0.05) in the muscle proximate composition was found between the white shrimp fed with dead probiotic cells and without probiotic cells. As for the muscle compositions of moisture, crude protein, and ash, there was no significant difference (P < 0.05) among all the groups (Table 3).

No significant difference (P > 0.05) was found in IMP contents between T-2 and control shrimp (Fig. 4). However, the white shrimp in the diet supplemented with viable probiotic cells showed a significantly greater (P < 0.05) IMP content (2.45 [+ or -] 0.11 mg/g) than T-2 and control shrimp (2.09 [+ or -] 0.12 mg/g and 1.97 [+ or -] 0.13 mg/g, respectively).


As with other aquatic animals, white shrimp have a close relationship with microorganisms. Thus, the use of probiotics for equilibrating their intestinal microflora, and the optimization of intestinal functions have received attention to increase the growth rates and welfare of farmed shrimp (Ziaei-Nejad et al. 2006, Castex et al. 2009, Yang et al. 2010). The current study showed that both viable and dead probiotic treatments improved the growth performance and survival rate of white shrimp compared with control shrimp, confirming findings of a previous study (Rachmilewitz et al. 2004). A similar observation was also made by Ghosh et al. (2003) in Indian carp (Labeo rohita) using the probiotic Bacillus circulans, which was isolated from fish gut. The effect of Bacillus spp. bacteria used as a probiotic on growth and survival has been demonstrated in the Indian white shrimp, Fenneropenaeus indicus (Ziaei-Nejad et al. 2006), and other shrimp species (Rengpipat et al. 1998, Gullian et al. 2004, Wang et al. 2005, Wang 2007, Liu et al. 2009, Liu et al. 2010, Javadi et al. 2011), although other studies found no positive effects (Shariff et al. 2001, Alavandi et al. 2004). In addition, the degree of probiotic benefit was different according to the form of probiotic application in the current study. Greater final weight and daily weight gain (and relative gain rate) were observed in the shrimp fed with live probiotic than in those supplemented with dead probiotic cells. This growth performance showed that the ingestion of probiotics might indeed modulate the white shrimp metabolism and immune system differentially. The above also indicated that the DNA of B. coagulans was only one of the factors promoting the beneficial effects in white shrimp. The growth and metabolism of probiotics, the interaction between probiotics and intestinal microflora, shrimp biological and chemical responses induced by exogenous probiotics, and environmental conditions were all important in the results of the probiotics trials. No significant difference was found in survival rate between T-1 and T-2, although there was a tendency for the rate to increase in T-1 shrimp. The results of survival rate suggested that partial beneficial biological effects, associated directly or indirectly with survival rate, obtained from viable cells of probiotics were also obtained from dead cells. A similar result was also found in crude fat, and there was also no significant difference between T-1 and T-2 shrimp. Indeed, the action of probiotics could be a dual one. According to Adams (2010), live probiotic cells influenced both the gastrointestinal microflora and the immune response whereas the components of dead cells exerted an immune response in the gastrointestinal tract. However, the molecular mechanisms by which viable and dead probiotics exert their beneficial effects have not been identified.

According to a previous study (Zhou et al. 2009), the use of viable B. coagulans in white shrimp (L. vannamei) larvae as a water additive improved digestive enzyme activity significantly. The current study showed similar results. Bacteria, particularly members of the genus Bacillus, can secret a wide range of exoenzymes (Moriarty 1998). Therefore, regarding the effects of probiotics in aquaculture, the nutrition enhancement of the host species through the production of supplemental digestive enzymes was one of the important modes of action (Verschuere et al. 2000). The significant increase in the specific activities of protease, amylase, and lipase of white shrimp treated with viable probiotics may enhance digestion and increase absorption of diet, which in turn contributes to better growth performance. Although we cannot distinguish the benefits resulting from enzymes synthesized by the shrimp or the bacteria, the exogenous enzymes produced by viable B. coagulans represent a contribution to the total enzyme activity of the gut, and the presence of the viable probiotic might stimulate the production of endogenous enzymes by the shrimp. However, no significant differences were observed in digestive enzyme activity between T-2 shrimp and control shrimp, suggesting that the better growth and survival rate in the shrimp with dead probiotics could be attributed to other probiotic beneficial modes of action, such as enhancement of the immune response of the host species (Panigrahi et al. 2005).

The data on the crude fat of white shrimp muscle in the current study indicated that probiotic effects were different according to the form of the probiotics. Because shrimp meat quality is associated with fat metabolism, a possible change in muscle composition and fat content could be expected. In the current study, our results showed that a lower crude fat content was obtained in T-1 shrimp than in the control shrimp, which suggests that the use of live probiotic cells was more effective. Lin et al. (2008) investigated the effect of viable probiotic Bacillus sp., supplemented in a diet with different concentrations, on the muscle proximate composition of white shrimp (L. vannamei)juveniles, and showed that the addition of probiotic strains at 1.0 g/kg improved crude fat content significantly compared with the groups treated with a probiotic at 0.5 g/kg and 2.5 g/kg. This result might be explained by the different probiotic strains and concentrations used. In addition, the result indicated that an additional amount of probiotic cells might not decrease further the fat content of shrimp muscle.

IMP, derived autolytically from the adenosine-5'-triphosphate (ATP) of the live tissue, was the main umami compound in the meat of poultry, livestock, fish, and shrimp, and played an important role in meat flavor formation (Kawai et al. 2002). As shown in Figure 4, a greater IMP content was found in shrimp fed the viable probiotic than in T-2 and control shrimp. There should be a positive correlation between the concentration of inosinic acid and ATP hydrolase activity, as well as a negative correlation between the concentration of inosinic acid and its degrading enzyme activity in muscle, because inosinic acid is the intermediate product in ATP metabolism. Therefore, the possible reason for the increased concentration of inosinic acid in white shrimp muscle is that the dietary viable probiotic cells influenced the system of ATP hydrolase and inosinic acid degrading enzymes. However, there was no significant difference in IMP content between T-2 and control shrimp in the current study. It is evident that the dead probiotic cells and extracellular products had no direct relation with the IMP metabolism system. Lee et al. (2001) and Cen et al. (2008) have demonstrated the close relationship between inosinic acid and meat in shrimp. Therefore, the addition of viable probiotic cells would have positive impact on the quality of white shrimp through an increased content of IMP.

In conclusion, dietary supplementation of both viable and dead probiotic B. coagulans appeared to be a promising practice for improve growth performance and survival rate of white shrimp L. vannamei. In addition, the probiotic effects were different according to the form of probiotic, and better values were observed in the group treated with viable probiotic cells. Further research is needed to understand more completely the functional mechanism of how the viable and dead probiotic cells work in the digestive tract of shrimp.


This study was supported by the Zhejiang Provincial Natural Science Foundation of China (no. R3110345) and National Natural Science Foundation of China (NNSFC, no. 31072221). We thank Dr. L. Hua for help with diet analysis.


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(1) Marine Resources and Nutrition Biology Research Center, Food Quality & Safety Department, Zhejiang Gongshang University, 149, Jiaogong Road, Hangzhou 310035, China; (2) Department of Biological Sciences, Florida Institute of Technology, 150 W. University Blvd., Melbourne, FL 32901

* Corresponding author. E-mail:

DOI: 10.2983/035.031.0333

Formulation and proximate composition of the basal diet.

Ingredients                  %     Proximate Composition   % Wet Weight

Fish meal                    38    Crude protein              40.54
Soybean meal                 15    Crude fat                   5.85
Peanut meal                  9     Ash                        13.72
Yeast                        5     Moisture                    8.11
Wheat flour                  20    Gross energy (MJ/kg)       16.94
Corn flour                   5
Menhaden fish                1
Soy oil                      1
Soy lecithin                 2
Mineral premix *             2
Vitamin premix ([dagger])    2

* Mineral premix to supply the following elements: zinc (as
sulfate), 72 mg/kg diet; iron (as sulfate), 36 mg/kg diet;
manganese (as sulfate), 12 mg/kg diet; copper (as sulfate), 24
mg/kg diet; cobalt (as chloride), 0.6 mg/kg diet; iodine (as
iodate), 1.2 mg/kg diet; chromium (trivalent, as chloride), 0.8
mg/kg diet; selenium (as selenate), 0.2 mg/kg diet; and
molybdenum (as molybdate), 0.2 mg/kg diet.

([dagger]) Vitamin premix: vitamin [B.sub.12], 0.1 mg/kg;
nicotinic acid, 80.0 mg/kg; riboflavin, 50 mg/kg; pantothenic
acid, 180 mg/kg; menadione, 40 mg/kg; folic acid, 6.0 mg/kg;
biotin, 0.6 mg/kg; thiamin hydrochloride, 15 mg/kg; pyridoxine,
60 mg/kg; thiamin, 40 mg/kg; inositol, 400 mg/kg; astaxanthin, 60
mg/kg; choline chloride, 20.0 mg/kg; vitamin C, 250 mg/kg;
vitamin A, 6,000 IU; vitamin [D.sub.3], 2,000 IU; and vitamin E,
6,000 IU.

Dietary probiotic, Bacillus coagulans, treatments on
growth performance and survival rate of white shrimp,
Litopenaeus vannamei.

                            Control                 Treatment 1

Initial weight (g)   0.85 [+ or -] 0.02        0.87 [+ or -] 0.03
Final weight (g)     5.58 [+ or -] 0.14 (a)    6.42 [+ or -] 0.13 (c)
Daily weight        0.094 [+ or -] 0.003 (a)  0.111 [+ or -] 0.002 (c)
  gain (g/day)
Relative gain rate   5.54 [+ or -] 0.25 (a)    6.41 [+ or -] 0.13 (c)
Survival rate (%)   76.22 [+ or -] 2.70 (a)   87.11 [+ or -] 3.42 (b)

                          Treatment 2

Initial weight (g)   0.86 [+ or -] 0.02
Final weight (g)     6.06 [+ or -] 0.11 (b)
Daily weight        0.104 [+ or -] 0.002 (b)
  gain (g/day)
Relative gain rate   6.07 [+ or -] 0.13 (b)
Survival rate (%)   82.22 [+ or -] 1.39 (b)

were significantly different (p < 0.05). Treatment I and
treatment 2 were fed a basal diet supplemented with viable and
dead probiotic cells, respectively; Control, fed basal diet only.
Means in the same row with different letters were significantly
different (p < 0.05).

Dietary probiotic, Bacillus coagulans, treatments on the
muscle proximate composition (%) of white shrimp,
Litopenaeus vannamei.

                         Control                   Treatment 1

Moisture        76.98 [+ or -] 1.64        75.53 [+ or -] 0.79
Crude protein   18.63 [+ or -] 1.83        19.82 [+ or -] 1.77
Crude fat        2.34 [+ or -] 0.11 (a)     1.94 [+ or -] 0.12 (b)
Ash              1.37 [+ or -] 0.13         1.46 [+ or -] 0.11

                       Treatment 2

Moisture        76.26 [+ or -] 1.38
Crude protein   18.38 [+ or -] 2.13
Crude fat        2.15 [+ or -] 0.12 (a,b)
Ash              1.42 [+ or -] 0.13

Treatment 1 and treatment 2 were fed a basal diet supplemented with
viable and dead probiotic cells, respectively; Control, fed basal diet
only. Means in the same row with different letters were significantly
different (P < 0.05).
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Article Details
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Author:Wang, Yanbo; Fu, Linglin; Lin, Junda
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:9CHIN
Date:Aug 1, 2012
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