LI Yan, LU Chunyu, YU Zhanqiao, and MA Qingshan, *

Isolation ofwith Feeding Attractant Function from Pacific White Shrimp () Intestine

LI Yan1), #, LU Chunyu2), #, YU Zhanqiao3), and MA Qingshan3), *

1),,266003,2),,571100,3),,266003,

In the present study, we isolated the lactic acid bacterium strain SC-01 from Pacific white shrimp () intestine. Using conventional and molecular methods, we identified the bacterium as, and found it had the function of feeding attractant and could inhibit the development of(zone of inhibition: 14mm). The attractant effect of itsfermentation broth is significantly better than that of the chemical attractant trimethylamine oxide (TMAO) (<0.05), and is equivalent to that of dimethyl-beta-propiothetin (DMPT) based on the feeding behavior of shrimp. High performance liquid chromatography (HPLC) analysis suggested that inosine-5’-monophosphate (IMP) may be a component of the attractant. A biosecurity evaluation revealed a negative result in hemolytic assays, and no shrimp mortality was resulted from SC-01 fermentation broth challenge. Feeding trials (60 days) indicated that the SC-01 fermentation broth (viable counts: 5.7×109cfumL?1) could improve feed intake, weight gain rate (WGR) and specific growth rate (SGR), and decrease the count ofsp. in the intestine of shrimp.

lactic acid bacterium;; probiotics; attractant;

1 Introduction

The rapid development of aquaculture industries in Chi- na has led to the shortage of feed resources and environment pollution, whichhas beena major problem to be solved. In addition, the demand for compound aquatic feeds is rising because of high-density farming techniques (Liu., 2017). However, concomitant growth in the use of compound aquatic feeds easily results in poor growth per- formance and low feed utilization efficiency, mainly because of the low palatability and attractiveness (Nunes., 2006; Qiu and Davis, 2018). The use of dietary feed- ing attractants in aqua feeds has, therefore, received considerable attentions in recent years. A small quantity of feed- ing attractant can improve feed intake and, as a consequence, increase the animal’s growth and survival (Tus- che., 2013; Olsén and Lundh, 2016; Kim and Cho, 2019).

Previous studies have demonstrated that various feed attractants including amino acids and their mixtures, sulfur- containing organic compounds, alkaloids and nucleic acids have high attractiveness for aquatic animals (Nunes., 2006; Silva-Neto., 2012). Most of these attractants are isolated from different marine organisms, such as shrimp, worms, krill, squid and mussels (Murai., 1983; Daniel and Bayer, 1987; Kolkovski., 2010; Alam., 2012). To our knowledge, attractants obtainedtho- rough fermentation of lactic acid bacteria (LAB) are rarely reported.

Lactic acid bacteria represent the majority of probiotics used in aquaculture (Gatesoupe, 2008). Species such as,,,, andare commonly used in aquaculture due to their positive effects (Ring? and Gatesoupe, 1998; Sun., 2012; Xing., 2013; Zhang., 2013; Zheng., 2017). In particular, the application ofto aquatic organisms has yielded beneficial effects, mainly in improving growth performance and reducing the mortality rate by inhibiting pathogens, regulating intestinal flora and stimulating the immune system (Bogut., 2000; Swain., 2009; Avella., 2011). These benefits may result from its ability to produce organic acids, exopolysa- ccharides and functional oligopeptides such as bacteriocin (Herranz., 2001; Abdhul., 2014; Abdel-Rahman., 2015). It has also been reported that LAB fermentation may produce flavor substances such as amino acids, flavor peptides, organic acids, aldehydes and ketones (Han., 2007; Coolbear., 2011). They may become attractants and thus improve the palatability of the feed. However, the attractant effect ofLAB, especially that of, on aquatic animalshas not been thoroughly investigated. The main aim of this study was to isolatefrom the shrimp intestine and evaluate the attractant effects of its fermentation broth on shrimp ().

2 Materials and Methods

2.1 Culture Media

To prepare screening medium, 0.75% CaCO3was added to the Man Rogosa Sharpe (MRS) agar medium. MRS, 2216E and thiosulfate citrate bile salts sucrose (TCBS) me-dium were purchased from Beijing Luqiao Technology Co., Ltd. (Beijing, China).

2.2 Sampling and LAB Isolation

Fresh shrimp () individuals were collected from high intensive shrimp culture ponds in Jiangsu, Fujian and Guangdong Provinces, China, respectively. The samples were stored in the ice box during transportation to laboratory, and analyzedat the same day. Shrimp body surfaces were washed and sterilized with 70% ethanol. Thenthe whole intestine, together with the contents, were sampled and homogenized in sterile saline (1:9, w/v) with a sterile glass homogenizer on ice. The homogenates were serially diluted in sterile saline (from 10?1to 10?6), and 100μL of the diluted homogenate was spread onto triplicate plates of screening medium. The inoculated plates were cultured at 37℃ for 48h in an incubator sett at a constant temperature (Yiheng, Shanghai, China). After incubation, colonies in the form of clear transparent circles with smooth surfaces and a diameter of 0.5–1mm were selected and streaked onto MRS agar to obtain the isolates. The isolates were subjected to safety evaluation using the hemolytic assays (Bernheimer, 1988). Each single colony was placed on the blood agar plate (Merck, Germany) and incubated at 37℃ for 24h, 48h and 72h for hemolysis observation. Purified strains (without hemolysis) were suspended in MRS broth containing 20% (v/v) sterile glycerol and stored at ?80℃for further analysis in the future.

2.3 Antimicrobial Activity Assays

The antibacterial tests were carried out using a simple filter paper (Gould, 1952).(la- boratory preservation) were used as the indicator,which was isolated and identified from diseased shrimp during the early mortality syndrome (EMS) outbreaks in the high intensive shrimp culture ponds in Guangdong Province, China. A dilution of thewas spread on TCBS plates. After impregnated and saturated with 50μL putative LAB fermentation broth (MRS medium at 37℃for 20h), paper discs (7mm in diameter) were placed on the agar plates. Plates were incubated at 28℃ for 24h, and the strains with obvious zone of inhibition were screen- ed. All tests were performed in triplicate.

2.4 Assessment of the Attractant Effect of Different Strains

Shrimps() were obtained from Charoen Pok- phand Group (Hainan, China) for the experiments. The healthy shrimps (8g±1.00g) were pre-incubated in tanks containing aerated sea water (salinity 20, pH 7.2) at 26℃ for 1 week for acclimation. The animal care protocol in this study was approved by the Institutional Animal Care and Use Committee of Ocean University of China. The attractant effect of different strains was evaluated using a pellet test as described by Cai and Ye (2005) and Hidaka. (2000) with some modifications.

The LAB fermentation broth was prepared using the following method. Fifty microliters of LAB strain storage solution was inoculated into 5mL MRS broth, and statically cultured at 37℃ for 20h in an incubator set at a constant temperature. Then the pellets were prepared using a mixture composed of 35% flour (crude protein: 9%– 11%, Luwang Group, Jining, China), 25% gluten (keep the insoluble pellets, crude protein >75%, Baipin biotech- nology, Xingtai, China), 39% water and 1% LAB strain fer- mentation broth.The mixture with different materials waspressed into individual pellets with a size of 0.5cm×0.3cmand a weight of 30mg. The shrimps were fed basal pellets (without attractant) for three days to acclimate to eat the pellets. Twenty pellets supplemented with LAB strain fermentation broth were placed in the same position in an indoor tank (50cm×40cm×50cm) containing 10 healthy shrimps.The pellets remaining were countedafter 10min. Pellets with 1% (v/w) MRS broth served as the control. The trial was repeated three times, and the status of the shrimp was consistent each trial (3h fasted). Ten LAB strains (named L1, L2, L3, L4, L5, L6, L7, L8, L9 and SC-01) were isolated fromintestine.The attractant index was calculated as follows:

2.5 Identification of Strain SC-01

Initial identification schemes were performed with bio- chemical tests as suggested by the Bergey’s Manual of Determinative Bacteriology (Holt, 1994). The molecular taxonomy of the isolates was determined by 16S rRNA gene sequencing. The total DNA was extracted using the DNA Extraction Kit (Sangon Biotech, Shanghai, China). The 16S rRNA gene fragment (V3V4 region) was amplified by polymerase chain reaction (PCR) using bacterial universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’), andsequenced by Shanghai Sangon Biotech Co., Ltd. (Shang- hai, China). The partial 16S rRNA gene sequence was com- pared with other bacterial sequence data using the Basic Local Alignment Search Tool (BLAST). A phylogenetic tree of strain SC-01 was constructed based on the comparison of the sequences with those of other referencebac- teria through Neighbor-Joining (NJ) analysis with 1000 boot- strap replicates (Molecular Evolutionary Genetics Analysis, MEGA 7.0 version).

2.6 Assessing the Biosecurity of Strain SC-01 for Shrimp

Shrimps () were obtained from Charoen Pok-phand Group (Hainan, China). The healthy shrimps (1.5cm±0.1cm) were pre-incubated in tanks containing aerated sea water (salinity 20, pH 7.2) at 26℃ for 1 week for ac- climation. To assay the safety of strain SC-01, 90 shrimps with similar body weight were randomly assigned to 3groups, 3 replicate tanks each group, 10 shrimps each tank (28cm ×18cm×17cm), in a recirculating system. Three treat- ment groups included: 1) control, without SC-01; 2) 104, with SC-01 at a concentration of 104cfumL?1; 3) 106, with SC-01 at a concentration of 106cfumL?1. During the ex- periment, half of the water in each tank was replaced with the fresh and filtrated sea water daily and shrimps were fed with the commercial pellet feed (Charoen Pokphand Group, Hainan, China) daily. Shrimp mortality was record- ed periodically at 24h, 48h, 72h and 96h after the expo- sure, respectively. The mortality rate was measured at the end of cultivation.

Mortality rate was calculated using the following equations:

2.7 Comparative Analysis of the Attractant Effect of Strain SC-01 and Other Ingredients

The attractant effects of strain SC-01 fermentation broth and other ingredients were evaluated based on the above method with some modifications, and different ingredi- ents were tested simultaneously in each tank (100cm×40cm×50cm). In addition, the preparation method of SC-01 fermentation broth was similar with that of LAB fermen- tation broth. Pellets were prepared using a mixture com- posed of flour, gluten, water and different attractant agents (a weight ratio of 1%, marked with different food color- ings; Wilton, Chicago, USA), andwe found that there was no difference on the number of remaining pellets of dif- ferent colors. Sixty pellets derived from three attractant agents of different colors (20 pellets each color) and 20 pellets from the control group for each test were placed in the same position in a tank containing 40 healthy shrimps (8g±1.00g). In test 1, SC-01 fermentation broth, MRS me- dium, peptone and control were analyzed.In test 2, SC-01 fermentation broth, acetic acid, lactic acid (Sinopharm Chemical Reagent, Beijing, China) andcontrol were ana- lyzed.In test 3, SC-01 fermentation broth, trimethylamine oxide (TMAO) (purity>99%, Zhonghong Biotechnology, Wuhan, China), dimethyl-beta-propiothetin (DMPT) (pu- rity>98%, DE Kay Biotechnology, Wuhan, China) and con- trol were analyzed. Pellets without supplement served as the control. The pellets remaining were counted after 10min. The trial was repeated three times, and the state of the shrimp used in each experiment was consistent (3h fasted). The attractant index was calculated using the equation below:

2.8 Analysis of SC-01 Fermentation Broth Components by RP-HPLC

Analysis of lactic acid, acetic acid and inosine-5’-mo- nophosphate (IMP) in SC-01 fermentation broth by LC3000 chromatograph (Luchuang Analytical Instrument, Shan- dong, China). Standards of the lactic acid, acetic acid and IMP (purity ≥99.8%) were purchased from Sigma (Sigma Chemical, St Louis, USA). Other chemicals (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All chemicals were of analytical grade except methanol and acetonitrile (HPLC grade).

Chromatographic analysis was performed using an ana- lytical Knaur C18 reversed phase column (150mm×4.6mm I. D.) with a particle size of 5.0μm (Agilent Tech- nologies, Palo Alto, USA). The mobile phase was 0.01molL?1phosphate buffer solution (pH 2.6) for analysis of lactic acid and acetic acid, and the methanol and phos- phoric acid (2:98, v/v) buffer solution (containing 0.005molL?1tetrabutyl bromoethane, pH 6.5) for analyzing IMP at a flow rate 1.0mLmin?1. The injection volume of each sample was 2μL. The column was thermostated at 30℃ for analyzing lactic acid and acetic acid and 50℃ for ana- lyzing IMP. All samples were detected at 210nm. Standard stock solutions of lactic acid (1.992gL?1), acetic acid (2.432gL?1) and IMP (6.1767gL?1) were prepared in ul- trapure water. Working solutions were prepared by dilut- ing the stock solutions with ultrapure water. The linear re- gression equation of the three analytes was described in Table 1. SC-01 fermentation broth was diluted with deio- nized water and filtered by a 0.45μm cellulose acetate mem- brane, and injected into the chromatograph and repeated 3 times.

Table 1 Linear regression equation for three analytes

Notes:, concentration (gL?1);, peak area;2, determination coefficient; IMP, inosine-5’-monophosphate. The RSD (relative standard deviation,=6 for each concentration) of these analysis methods was <2%.

2.9 Effects of SC-01 Fermentation Broth on Growth Performance and Intestinal Flora of Shrimp

Healthy shrimps() were obtained from Cha- roen Pokphand Group (Hainan, China) and were accli- mated at 26℃±1℃ and a salinity of 20 in tanks, fed the basal diet three times a day for 1 week. The basal feed was purchased from Charoen Pokphand Group (commer- cial shrimp feed, Hainan, China).The nutrient contentsin-clude crude protein 42.85%, ether extract 8.41%, mois- ture 8.53%, ash 10.16%, Ca 1.94% and total phosphorus 1.02%.

To prepare the SC-01 fermentation broth, 100μL ofSC-01 storage solution was inoculated in 10mL MRS broth at 37℃. After 20h, it was inoculated into 1L fresh MRS broth, and statically incubated for another 20h at 37℃. After incubation, the SC-01 fermentation broth was harvested and stored at 4℃, and the viable counts of SC-01 fermentation broth (5.7×109cfumL?1) were deter- mined by plating on MRS agar.

Feeding experiment was conducted in 12 indoor tanks (2.0m×1.2m×1.0m) in Pearl River Fisheries Research Institute, Guangzhou, China. Six hundred shrimps with an initial average weight of 5.55g±0.20g were randomly assigned to three groups, 50 each tank, 4 tanks each group. The diets in three treatment groups included the basal diet without SC-01 fermentation broth (control); the supple- mented with 1% (v/w) SC-01 fermentation broth in the basal diet (SC-01 (1%)); the supplemented with 2% (v/w) SC-01 fermentation broth in the basal diet (SC-01 (2%)). The basal diet and SC-01 fermentation broth were mixed immediately before feeding. Shrimps were fed four times a day at 5% of body weight at 06:00, 12:00, 17:00 and 23:00 for 60 days, and uneaten feed was collected after 1h of feeding and then immediately dried. The feed intake was calculated by subtracting the uneaten portion and recorded daily. Throughout the experimental period, tem- perature was ranged from 25℃ to 27℃, pH was 7.5–8.5, dissolved oxygen content was approximately 6.0mgL?1, and total ammonia nitrogen content was <0.2mgL?1.

At the end of the experiment, shrimps were fasted for 24h and then weighed. Three shrimps from each tank were randomly selected. Their whole intestines were aseptically dissected using sterilized surgical scissors, and immedi- ately homogenized for counting the number of intestinal bacteria. The counts of the total culturable bacteria (TCB), LAB andsp. in intestines were conducted as de- scribed by Zhang. (2010) using 2216E, MRS and TCBS agar plates, respectively. The weight gain rate (), specific growth rate (), feed conversion rate () and survival rate () were calculated as follows:

wheremeans final weight;means initial weight; andmeans total experimental days.

2.10 Statistical Analyses

All data were analyzed by one-way ANOVA using Sta- tistical Analysis Systems (SAS). Signi?cant difference was assessed at<0.05.

3 Results

3.1 Isolation of LAB Strains

Cultured on MRS plates at 37℃ for 48h, 10 isolates with clear transparent circles, smooth surfaces, hemolytic assays negative and round colony diameter 0.5–1mm were screened out. The results of re-screening of pellet test showed that the attractant index of SC-01 strain was sig- nificantly higher than that of other strains and control (Fig.1), thus, its attractant effect on shrimp was better than that of other strains.

Fig.1 Attractant index of 10 selected strains. Values are expressed as the mean (n=3) and the error bars represents standard deviation (SD). Means with different letters a, b, c are significantly different (P<0.05).

In the antibacterial test,, a com- mon pathogen in aquaculture, was selected as an indicator. The test result showed that the strain SC-01 had a sig- nificant inhibitory effect on(Table 2), and an obvious inhibition zone (diameter 14mm) was ob- served.

Table 2 Antibacterial activities of different strains (n=3)

Notes: Indicator,. +++ means inhibition zone≥12mm (significantinhibition); ++ means 12mm≥Inhibition zone≥8mm (inhibition); + means 8mm≥Inhibition zone≥7mm (contact inhibition); – means no antibacterial activity.

3.2 Identification of Strain SC-01

SC-01 showed the typical characteristics of LABspe- cies, which were gram-positive, catalase test-negative, VP test-positive, Arg production of ammonia test-positive, H2Stest-negative, gelatin test-negative, indole test-negative, and no spore-forming bacteria (Table 3). Strain SC-01 was also able to grow with 6.5% NaCl at 10–45℃ and pH 4.5–9.6, which suggests that this strain is suitable for using in ma- rine aquaculture.

The BLAST program was used to identify homologous sequence in GenBank (http://www.ncbi.nlm.nih.gov/). The MEGA7.0 was used to construct a phylogenetic tree as is shown in Fig.2. The 16S rRNA gene phylogenetic analy- sis revealed that the SC-01 strain shows 99% similarity toDSM 20477T(accession number: AJ276355.1). Based on the above results, the strain SC- 01 was identified as.

Table 3 Physiological and biochemical characteristics of strain SC-01

Notes:+ and – represent the positive and negative responses, re- spectively.?BS means Bile salts.

Fig.2 Phylogenetic tree based on 16S rRNA gene sequence of strain SC-01.

3.3 Biosecurity of Strain SC-01

SC-01 presented negative results in the hemolytic as- says (Fig.3). When the culture of SC-01 was added to shrimp sink, no shrimp death was observed in any test arrange- ment. Thus SC-01 is safe for aquaculture farming (Table 4).

Fig.3 Hemolytic analysis of strain SC-01.

Table 4 The effect of strain SC-01 on mortality of shrimp

3.4 Estimation of Attractant Effect of Different Ingredients

Comparison of the attractant effect of the strain SC-01 fermentation broth, medium components, organic acid and chemical attractants is shown in Fig.4. The attractant index of SC-01 group was significantly higher than that of MRS medium, peptone, acetic acid, lactic acid, TMAO and control group (<0.05) (Figs.4A, B and C), which had no significant difference with that of the DMPT group (Fig.4C). The result indicated that the fermentation broth of strain SC-01 has the potential to be developed into a biological attractant.

Fig.4 Attractant index of different attractant ingredients. Values are expressed as the mean (n=3) and the error bars represent standard deviation (SD). Means with different letters a, b, c are significantly different (P<0.05). MRS, Man Rogosa Sharpe medium; DMPT, dimethyl-beta-propiothetin; TMAO, trimethylamine oxide.

3.5 Determination of SC-01 Fermentation Broth Components

HPLC analysis of SC-01 fermentation broth composition is shown in Fig.5. The retention time of lactic acid, acetic acid and IMP were 3.952, 4.197 and 6.972min respec-tively (Figs.5A1, B1). The components of SC-01 fermen- tation broth including lactic acid, acetic acid and IMPwereconfirmed by comparing the chromatogram peaks between SC-01 fermentation broth and the standard solutions (Figs. 5A2, B2), with average contents of 5.79, 0.72 and 0.18gL?1, respectively.

Fig.5 HPLC chromatograms of a mixture of standard solutions of lactic acid and acetic acid (volume ratio 1:1) (A1), standard solutions of inosine-5’-monophosphate (IMP) (B1), SC-01 with standard solutions of lactic acid and acetic acid (A2), and SC-01 with standard solutions of IMP (B2). Peak 1, lactic acid; peak 2, acetic acid; and peak 3, IMP. SS, stan- dard solutions.

3.6 Growth Response

The effects of SC-01 fermentation broth on the growth performance are shown in Table 5. There was no signifi- cant difference in initial weight among three groups. The final weight in the two treatment groups was significantly higher than that in the control group (<0.05). The va- lues of, feed intake andin SC-01 (2%) treat- ment group were significantly higher than those in the con- trol group (<0.05), and these indicators showed a higher trend in the SC-01 (1%) group. No significant difference was recorded inandamong three groups (>0.05). These results indicated that SC-01 fermentation broth can improve growth performance of shrimps.

Table 5 Effects of SC-01 fermentation broth on growth performance of shrimp

Notes: Within a row, values with different superscripts are significantly different (<0.05). The values are shown as means±SD (=4). Means with different lettersa, b, care significantly different (<0.05)., weight gain rate;, specific growth rate;, feed conversion rate.

3.7 Intestinal Bacterial Counts of Shrimp

The result of the intestinal bacterial counts in the shrimp is shown in Fig.6. There was no significant difference in the number of total TCB in the intestines among three groups (>0.05). Compared with the control group, the two treatment groups significantly increased the count of LAB (<0.05) and decreased the count ofsp. (<0.05) in the intestine of shrimp.

Fig.6 The effect of SC-01 fermentation broth on total cul- turable bacteria (TCB), lactic acid bacteria (LAB) and Vi- brio sp. counts in the intestine of shrimp. Values are ex- pressed as the mean (n=4) and the error bars represent standard deviation (SD). Means with different letters a, b are significantly different (P<0.05).

4 Discussion

The results demonstrated that the LAB strain isolated from shrimp () intestine had antibacterial ac- tivity and attractant effect. The strain was identified asSC-01, based on morphological and biochemical features as well as on 16S rRNA gene sequence. Strain SC- 01 is able to grow with 6.5% NaCl at 10–45℃ and pH 4.5–9.6, which suggested that it may be suitable for ma- rine aquaculture. The biosecurity evaluation revealed nega- tive results in the hemolytic assays and no shrimp mortal- ity resulted from SC-01 fermentation broth challenge. There- fore, SC-01 is safe as a potent agent for feeding stimulants in aquatic farming.

The pellet test in this study is an improvement of the feeding behavior method and strain SC-01 showed the greatest attractant potential compared with other strains. This method does not require special equipment and has several favorable characteristics including simple opera- tion, short screening period and high accuracy (Cai and Ye, 2005). A previous study showed that moststrains have a good antibacterial activity, and are used as probiotics to antagonize cohesivein cli- nical medicine (Miyazaki., 2010). Furthermore,can also have a good inhibitory activity against com- mon aquatic pathogens such as,and(Sun., 2010), and protect shrimp () from attacks by(Swain., 2009). The present study revealed thatSC-01 had an inhibitory activity against. This suggests that strain SC-01, as a bioin- ducer, can promote shrimp feeding, inhibit pathogens and improve intestinal health.

The use of attractants can improve the palatability of feeds, enhance animal appetite and feed intake, reduce feed waste and improve growth performance in aquaculture (Pa-patryphon and Joseph, 2000; Pratoomyot., 2010). Attractant substances are mainly synthesized chemicals, or extracts from animals and plants. They comprise amino acids, alkaloids, sulfur compounds, Chinese herbal medi- cines, animal and plant extracts, and so on (Smith., 2005; Silva-Neto., 2012). Some attractants are ob- tained from microbial extracts, fermentation feeds and fer- mentation broths (Mato., 1977; Guerenstein., 1995). These attractants not only have attractant effects, but also have other functions, including regulating intes- tinal flora balance, improving host immunity, and reduc- ing intestinal diseases (Banerjee and Ray, 2017). Gueren- stein. (1995) reported that a volatile substance pro- duced byhas a high attractant effect in. In aquaculture, fermented soy- bean meal is the most researched fermented feed, which can significantly improve the food intake of aquatic ani- mals and enhance production performance (Rombenso., 2013; Azarm and Lee, 2014).

Previous studies have shown that probiotic strains ofhas been widely using in aquaculture mainly due to their excellent effects in inhibiting pathogens, regulat- ing intestinal flora and enhancing immunity (Bogut., 2000; Swain., 2009; Avella., 2011). However, the attractant effect of the fermentation broth ofis rarely reported. Our study revealed that the SC-01 fermentation broth can promote the feed intake of shrimp. Its attractant effect is significantly better than that of MRS medium, peptone, acetic acid, lactic acid and the attrac- tant TMAO, and is equivalent to that of the DMPT, thus it has the potential of being developed into a biological at- tractant. The similar result has been obtained in a previ- ous study showing that fermentation broths of yeast and sugar can be attractants to moths (Utrio and Eriksson, 1977).

Microbial fermentation broths and fermented raw ma- terials can promote the feed intake of aquatic animals. This may be partially due to the flavor substances (., amino acids, small peptides and nucleotides) produced during microbial fermentation (Han., 2007; Coolbear., 2011). In the present study, the attractant effect of SC-01 fermentation broth was significantly better than that of the MRS medium and peptone, suggesting that the attractant ingredients may be some metabolites produced by the microorganisms. The active components of attrac- tants can be assessed by carrying out an efficacy compa- rison test and a product detection test (Millar., 1992). Millar. (1992) analyzed a fermented Bermuda grass infusion by liquid chromatography and identified that the main attractant components included phenol, 4-methyl- phenol, 4-ethylphenol, indole and 3-methylindole. Another report showed that IMP can enhance the feeding activity of jack mackerel(Ikeda., 1988). In this study, a small amount of IMP was also detected by HPLC, indicating that this component may participate in the attractant effect to shrimp. However, LAB fermenta- tion can also produce small peptides, aldehydes, ketones and other flavor substances (Coolbear., 2011), which may also have important attractant effects. Further research is needed to reveal the components having attractant effi- cacy by means of liquid chromatograph-mass spectrome- ter (LC-MS) and gas chromatography-mass spectrometer (GC-MS).

Many studies have showed that ansupple- ment had many positive effects in aquatic animals, inclu- ding reducing the feed coefficient, promoting growth, in- hibiting pathogenic bacteria (such as,and), and improving im- munity and survival rate (Wang., 2008; Swain., 2009; Avella., 2011; Sun., 2012). In the present study, a higher final weight,and, and a sig- nificantly increased feed intake were observed in the treat- ment groups compared with the control group. This was particularly marked in the SC-01 (2%) group, which sug- gested that SC-01 fermentation broth can improve the growth performance and may partly contribute to its at- tractant effect. Similarly, Wang. (2008) reported thatZJ4 (1×107cfug?1) in feed significantly im- proved final weight and daily weight gain in tilapia. Bogut. (2000) reported that 2×108cfug?1ofsig- nificantly improved the daily weight gain (up to 10.85%) in sheat fish ().

It is well known that the gastrointestinal microflora of aquatic animals play an important role in the growth and health of the animals (Nayak, 2010). The components and amounts of gastrointestinal microbes in aquatic animals are easily affected by culture environment, supplementa- tion of feed and some additives (Zhang., 2011). Ge- nerally, probiotics supplements can exert beneficial ef- fects on the host by inhibiting pathogenic bacteria, im- proving the microbiological balance and maintaining in- testinal health(Banerjee and Ray, 2017). The present study showed that the number of detectedsp.was signi- ficantly decreased by feedingSC-01, which may be related to its antibacterial activity. Our findings agree with those of Adel. (2017) who found that a dietaryprobiotics significantly decreased the number ofsp. in the intestine of shrimp.

Our results indicated thatSC-01 fermenta- tion broth may inhibit, increase feed intake and improve the growth performance of shrimp (). Hence,SC-01 fermentation broth has the potential to be developed into a bioattractant. Fur- ther studies should be carried out using LC-MS and GC- MS to identify the possible attractant ingredients, and ana- lyze the relationship between attractant ingredients and improved growth performance.

Acknowledgement

This research was supported by the Special Fund for Qingdao Marine Biomedical Science and Technology In- novation Center, China (No. 2017-CXZX01-3-13).

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# These authors contributed equally to this work.

. E-mail: horsegreenhill@163.com

September 24, 2019;

November 13, 2019;

March 25, 2020

(Edited by Qiu Yantao)