EFFECT OF AQUEOUS MORINGA ( MORINGA OLEIFERA ) LEAF EXTRACT AS A PREBIOTIC ON GROWTH OF THE WHITELEG SHRIMP, PENAEUS VANNAMEI BOONE, 1931 (DECAPODA, PENAEIDAE)

This study evaluated the potential of the aqueous moringa ( Moringa oleifera ) leaf extract (ME) as a prebiotic and its effect on the growth performance of the whiteleg shrimp, Penaeus vannamei Boone, 1931. The growth trial of probiotics in the broth containing ME showed that Lactobacillus acidophilus growth rate was signiﬁcantly higher ( P< 0 . 05) than that of Lactobacillus plantarum and Lactobacillus reuteri . Furthermore, L. acidophilus exhibited also better growth ( P< 0 . 05) than Vibrio alginolyticus , Vibrio parahaemolyticus and Vibrio harveyi in the broth containing ME. The minimum inhibitory concentration of ME (MIC) and minimum bactericidal concentration (MBC) were the same against V. alginolyticus , V. parahaemolyticus , and V. harveyi at concentrations of 10, 7.5, and 7.5 mg/ml, respectively. By contrast, ME’s MIC and MBC against L. acidophilus were 50 and > 100 mg/ml, respectively. The prebiotic scores of L. acidophilus against V. parahaemolyticus and V. harveyi were signiﬁcantly higher ( P< 0 . 05) than that against V. alginolyticus . The MIC, MBC, and prebiotic scores indicated that ME enhanced the growth of L. acidophilus more than that of Vibrio bacteria. Six diets were formulated for the feeding trial; 3 diets contained ME at 0 (ME0), 2.5 (ME2.5) and 5.0 g/kg (ME5.0), respectively, and the same 3 diets were also prepared with L. acidophilus (ME0 + P, ME2.5 + P, and ME5.0 + P, respectively). After the feeding trial, the number of lactic acid bacteria in the gut was higher ( P< 0 . 05) in the ME2.5 + P group than in the control and ME2.5 groups, but no differences were found in the vibrio-like bacterial count among the treatments. The ME2.5 + P group had a higher ( P< 0 . 05) ﬁnal weight, weight gain, and speciﬁc growth rate than did the ME or L. acidophilus alone and the control groups. Therefore, ME exhibits a prebiotic function and exerts a symbiotic effect with L. acidophilus , thus increasing the growth performance of P. vannamei .


INTRODUCTION
The whiteleg shrimp, Penaeus vannamei Boone, 1931 (by some authors still referred to as Litopenaeus vannamei (Boone, 1931)), constitutes a commercially valuable shrimp and is, next to being caught in the wild, also raised in aquaculture for human consumption (Figueredo et al., 2023).As a consequence, aquaculturists, in particular in Southeast Asia, are constantly trying to improve culturing conditions of this species, in attempts to raise production and thus to enhance yields (Manan & Ikhwanuddin, 2021).One aspect of such improvements could be found in influencing the gut microbiota of P. vannamei, which may lead to better health conditions of the shrimp and thus enhance survival (El-Saadony et al., 2022).In the present study, the use of moringa for improving the gut microbiota of these shrimps has been investigated.
Moringa, Moringa oleifera Lamarck (Brassicales, Moringaceae), has many local names (including mother milk, drumstick tree, miracle tree) but its most common English name is horseradish tree.Moringa is originally from India and grown in subtropical and tropical regions worldwide (Foidl et al., 2002).Moringa grows rapidly, as much as 5 m/year (Morton, 1991), and is well known for its nutritional content, including 27.12% crude protein, 2.33% crude lipid, 19.17% fibre, 6.1% ash, and 36.88% nitrogen-free extract, vitamins and minerals (Sherif et al., 2014).Thus, different parts of moringa, including the pods, leaves, seeds, gums, barks and flowers, are widely consumed as food or used as traditional medicine (Mahmood et al., 2010).
The water extract of moringa leaves has antibacterial properties that inhibit the growth of some bacteria, including Escherichia coli (Migula, 1895) Castellani & Chalmers, 1919, Staphylococcus aureus Rosenbach, 1984, and Streptococcus pneumoniae (Klein, 1884) Chester, 1901(cf. Idris & Abubakar, 2016), but not that of other bacteria, such as Bacillus pumilus Meyer & Gottheil, 1901, Bacillus cereus Frankland & Frankland, 1887, and Streptococcus faecalis Andrewes & Horder, 1906(cf. Moyo et al., 2012).The actual antibacterial properties of moringa depend on the solvent used during the extraction process: the antibacterial efficacy of the acetone and methanol extracts of moringa leaves is higher than that of aqueous extracts (Moyo et al., 2012;Idris & Abubakar, 2016).However, the aqueous extract offers numerous advantages as a green extraction solvent, because water is inexpensive, nontoxic, environmentally friendly, and provides an opportunity for clean processing and preventing pollution (Filly et al., 2016).
Moringa might act as a prebiotic in diets (Gbadebo et al., 2019;Li et al., 2021;Prayitno et al., 2021).Prebiotics are non-digestible nutrients that stimulate the increase of beneficial gut microbiota in the host, whereas probiotics are live microbial feed additives that beneficially affect the host; therefore, the prebiotic supports probiotic growth (Akhter et al., 2015).Ordaz et al. (2018) found that phenolic compounds in the plants selectively promote probiotic growth and inhibit pathogenic bacterial growth.Furthermore, Elabd et al. (2018) reported that polyphenols in herbs can modify the gut microbial composition.
Depending on the extraction technique, the number of phenolic compounds in the moringa leaf extract may vary from 2.3 to 13.5% (Vongsak et al., 2013).Moreover, the polysaccharide extract of the moringa leaf modulates microbial composition in the intestines (Li et al., 2021).A study reported a decrease in the numbers of E. coli, Salmonella and Staphylococcus spp.but an increase in the number of Lactobacillus in the small intestine of broiler chickens, Gallus gallus (Linnaeus, 1758), fed with moringa (Hafsa et al., 2019).Moreover, moringa supplementation in the diet of mice (Mus musculus Linnaeus, 1758) affects the gut microbiota composition, increases the number of Lactobacillus, and reduces the number of Bifidobacteria (Elabd et al., 2018).Wang et al. (2019) demonstrated that the polysaccharide extract of the moringa leaf exerts a prebiotic effect by maintaining the integrity of the intestinal mucosa and modulating the gut microbiota composition.Therefore, moringa may function as a prebiotic in the whiteleg shrimp, Penaeus vannamei.
A plant is classified as a prebiotic if it selectively stimulates the growth of beneficial bacteria; consequently, intestinal microorganisms can ferment the prebiotic, and the prebiotic resists host digestion (Gibson et al., 2017).Thus, inclusion of moringa in the diet of whiteleg shrimp can promote the growth of beneficial bacteria, such as Lactobacillus, and reduce the growth of pathogenic bacteria, such as Vibrio.Prebiotic activity can be determined in vitro by comparing the growth of beneficial bacteria with that of enteric pathogens in the presence and absence of the given prebiotic in the medium (Mallik & Bhawsar, 2018).
Prebiotics affect not only the growth of beneficial bacteria but also the health and growth performance of animals (Ringo et al., 2010).For example, the addition of prebiotic honey in Penaeus vannamei diet increased their total weight and reduced the feed conversion ratio (Fuandila et al., 2020).Furthermore, supplementation of prebiotics and probiotics in the diet of P. vannamei resulted in improved growth compared with the use of a prebiotic or probiotic alone (Arisa et al., 2015).
To the best of our knowledge, no study has evaluated the antimicrobial and prebiotic activity of the aqueous moringa leaf extract (ME) against shrimp pathogenic bacteria.Therefore, this study evaluated the potential of the aqueous ME as a prebiotic by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), prebiotic score, as well as lactic acid bacterial count and vibrio-like count in the intestine.Additionally, this study investigated the effect of diet supplementation with the aqueous ME and Lactobacillus acidophilus (Moro, 1900) Hansen & Mocquot, 1970 on the growth of Penaeus vannamei.

ME preparation
Moringa oleifera leaves were purchased from Rumah Kelor, Blora, Indonesia.The leaves were prepared as previously described by Abidin et al. (2022b).The leaves were cleaned and air-dried for 3 days at 35°C.The dried leaves were grounded into a fine powder, mixed with distilled water (98°C) at a ratio of 1 : 9, and stored for 24 h at room temperature.The solution was filtered using a muslin cloth and Whatman Filter Paper No. 1 (Whatman, Brentford, U.K.) to separate the leaves from the liquid solution.The solution was then frozen at −80°C and dried in a freeze dryer for 3 days until the solution turned to powder.The powder was stored at −20°C until further use.

Bacterial growth in ME
To prepare the bacterial broths, we transferred 10 μl of each probiotic stock to 10 ml of MRS broth supplemented with the ME concentrations of 0, 0.25, 0.50, and 1.00 mg/ml.The probiotics were then incubated at 28°C for 24 h.The probiotic with the highest growth rate at low ME concentrations was selected for the next step of the experiment.The selected probiotic, namely Lactobacillus acidophilus, was then cultured in different ME concentrations (0,5,10,25,50,75, and 100 mg/ml) to determine the optimal ME concentration for bacterial growth.The concentration that resulted in the superior growth of L. acidophilus, namely 5 mg/l, was used in the growth test of Vibrio bacteria.
A total of 10 μl stock of each Vibrio and L. acidophilus was transferred to 10 ml of broth containing 5 mg/ml of the ME.The bacteria were incubated at 28°C for 48 h.Bacterial growth was measured at 6, 12, 24, and 48 h.
To count the bacteria, a bacterial solution of L. acidophilus (200 μl) and 200 μl of solutions of each Vibrio were spread on MRS and TSA agar, respectively, then and incubated for 24 h at 28°C.All the experiments were conducted in triplicate.

MIC and MBC tests
The MIC and MBC were determined using the broth dilution method previously described by Unegbu et al. (2020).The ME was dissolved in broth to obtain the ME concentrations of 0, 5, 7.5, 10, 25, 50, 75 and 100 mg/ml.Stock of each bacterium (L.acidophilus, V. parahaemolyticus, V. alginolyticus and V. harveyi; 10 μl each) was transferred into a 10-ml broth containing various ME concentrations.One of each ME concentration was not inoculated with bacteria and served as a negative control.All bacteria were incubated at 28°C for 24 h.The lowest ME concentration that indicated no visual turbidity change was considered as the MIC.
After the MIC was determined, 200 μl of bacteria from all the tubes with no visible bacterial growth were seeded on the MRS (for L. acidophilus) and TSA (for Vibrio bacteria) and incubated at 28°C for 24 h.The lowest ME concentration that exhibited no bacterial colony on the agar was considered as the MBC.In each case, three experiments were conducted in parallel.

Prebiotic activity score
The prebiotic activity score was determined using the method previously reported by (Thitiratsakul & Anprung, 2014) with some modifications.L. acidophilus was the representative probiotic, and Vibrio bacteria were the pathogenic species.A 10-μl bacterial volume was taken from the stock and inoculated in 10 ml of broth containing 5 mg/ml of ME or glucose for 24 h at 28°C.All the bacterial colonies grown on TSA at 28°C were counted.The prebiotic activity score was determined using the following equation:

Dietary ME preparation
Six experimental diets were formulated.The diets contained either both ME and L. acidophilus or only ME or L. acidophilus alone (table I).Three diets contained ME at 0, 2.5, and 5.0 g/kg, which were denoted as ME0, ME2.5, and ME5.0, respectively.The same 3 diets were sprayed with L. acidophilus at a concentration of 1 × 10 7 CFU/g diet, which were denoted as ME0 + P, ME2.5 + P, and ME5.0 + P, respectively.These diets were dried for 24 h at 35°C and stored at 4°C until further use.The proximate compositions of the diet (i.e., lipid, crude protein, ash, and moisture) were analysed using standard methods (Helrich, 1990).

Lactic acid and Vibrio-like bacterial count in the gut
Eighteen healthy shrimp, Penaeus vannamei (14.5 ± 0.3 g) were randomly distributed into six 75-litre tanks.The shrimp were fed with the experimental diets for 7 days.On the final day, the shrimp were starved for 12 h and dissected to retrieve and weigh the whole gut, which included the foregut (stomach), midgut and hindgut.The whole gut was homogenized in phosphate-buffered saline at a ratio of 1 : 9 (w/v).To count lactic acid and Vibrio-like bacteria, 200 μl of the homogenized gut was spread on MRS and TCBS agar, respectively, and incubated at 28°C for 48 and 24 h, respectively.The colonies that appeared on the agar were evaluated.

Growth performance
In total, 180 juvenile whiteleg shrimp (2.00 ± 0.25 g) were randomly distributed into eighteen 75-litre tanks (6 groups with 3 replications per group).The shrimp were reared in a recirculation system for 30 days and fed 3 times a day with the experimental diet at 5% body weight.The shrimp were weighed on day 14, and the amount of feed was adjusted accordingly.Water was maintained at the following conditions: a temperature of 28-29°C, oxygen >6 mg/l, salinity of 30 to 33 , and pH of 6.8 to 7.6.The growth parameters were measured on the final day by using

Statistical analysis
The experiments were arranged in a randomized set.Shapiro-Wilk and Levene's tests were used to test the normality and homogeneity of the data before data were subjected to one-way analysis of variance (ANOVA).Duncan's test was used to determine significant differences among the treatments.A P value of <0.05 was considered statistically significant.All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, U.S.A.).

Bacterial growth in ME
Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus acidophilus were grown in ME at 28°C for 24 h.The growth of L. acidophilus was significantly higher (P <0.05) than that of the other bacteria in the aqueous ME at a concentration of 0.25 mg/ml.Additionally, at the ME concentrations of 0. 50 and 1.00 mg/l, L. plantarum and L. acidophilus exhibited significantly better growth (P <0.05) than did L. reuteri (P <0.05; fig.1).Therefore, L. acidophilus was used as the probiotic in the present study because it demonstrated the highest growth performance even at a low ME concentration.
An ME concentration of 5 to 25 mg/l resulted in better growth (P <0.05) than did ME concentrations of 0 and of 75 to 100 mg/l (fig.2).The growth of L. acidophilus tended to decrease at ME concentrations above 25 mg/l.Fig. 3 shows that the growth of L. acidophilus was significantly lower (P <0.05) than that of the other three Vibrio bacteria at 6 and 12 h (P <0.05).However, at 24 h, the growth of L. acidophilus was higher (P <0.05) than that of both Vibrio harveyi and Vibrio parahaemolyticus but lower than that of Vibrio alginolyticus (P <0.05).At 48 h, the ME resulted in higher growth (P <0.05) of L. acidophilus compared with the three Vibrio bacteria.

MIC and MBC
As shown in table II, the MIC of ME for L. acidophilus was 50 mg/ml, and the bacterium remained alive even at an ME concentration of 100 mg/ml.However, the MIC and MBC values were the same at the ME concentrations of 10.0, 7.5 and 7.5 mg/l for V. alginolyticus, V. parahaemolyticus and V. harveyi, respectively.et al., 1961) Sakazaki, 1968, Vibrio parahaemolyticus (Fujino et al., 1951) Sakazaki et al., 1963), and Vibrio harveyi (Johnson & Shunk, 1936) Baumann et al., 1981.Therefore, ME inhibited the growth of Vibrio more effectively than that of L. acidophilus.
acidophilus, either individually or in a combined diet, did not affect the total number of Vibrio-like bacteria in the gut of Penaeus vannamei (fig.4b).

Growth performance
The final weight, weight gain, and SGR were significantly higher (P <0.05) in the ME2.5 + P group than in the other groups (table IV).No differences in growth performance parameters were observed between the ME5.0 and ME5.0 + P groups.The ME0 + P group exhibited a higher final weight, weight gain, and SGR than did the ME0 group.Furthermore, weight gain and SGR did not significantly differ between the ME2.5 + P and ME5.0 + P groups.No significant difference in the FCR was noted among all the treatment groups; however, all treatment groups had considerably higher FCRs than did the control group (P <0.05).

DISCUSSION
Prebiotics are mainly derived from disaccharides and simple polysaccharides (Yoo et al., 2012).For instance, polysaccharides from mushrooms promote the growth of Lactobacillus (Nowak et al., 2018).Prebiotics are commonly associated with nondigestible carbohydrate fibres; however, in the last 10 years, the polyphenolic compound has garnered attention as a potential prebiotic (Chen et al., 2015).Furthermore, Milutinović et al. (2021) reported that polyphenols are poorly digestible and considered a novel prebiotic group.The aqueous ME contains approximately 6.84% polysaccharides (Li et al., 2020) and has a high total phenolic content of 2.35 to 4.41%.
The present study revealed that Lactobacillus acidophilus and Lactobacillus plantarum demonstrated higher growth performance among the three Lactobacillus tested.One study revealed that various Lactobacillus species exhibit different growth responses even if they are cultured in the same herb extract (Milutinović et al., 2021).Moreover, different herb extracts yield different growth rates of Lactobacillus (Nanasombat et al., 2018).The growth performance of L. acidophilus is higher than that of Streptococcus thermophilus Orla-Jensen, 1919and Bifidobacterium animalis (Mitsuoka, 1969) Scardovi & Trovatelli, 1974emend. Masco et al., 2004 in banana (Musa acuminata Colla) peel fibre powder (Santo et al., 2012).Additionally, mangosteen extract (Garcinia mangostana Linnaeus, 1753) resulted in a higher growth performance of L. acidophilus than did gac extract (Momordica cochinchinensis (Lour.)Spreng.)(Nanasombat et al., 2018).The results of the present study indicate that ME as an aqueous solution has prebiotic potential and is suitable for enhancing the growth of L. acidophilus.
An ME concentration of 5 to 25 mg/ml yielded the maximum growth of L. acidophilus.A mangosteen, nutgrass (Cyperus rotundus Linnaeus, 1753), and gac extract concentration of 5 mg/l increased the growth of L. acidophilus (Nanasombat et al., 2018).The ME extract did not inhibit the growth of Vibrio parahaemolyticus and V. alginolyticus after 24 h of incubation.However, the growth of the two Vibrio bacteria decreased at 48 h, and the growth of L. acidophilus increased.Compared with the growth of other Vibrio bacteria, the growth of V. harveyi was more strongly inhibited by ME.
We determined the same value of the MIC and MBC for V. alginolyticus, V. parahaemolyticus and V. harveyi at the ME concentrations of 10.0, 7.5 and 7.5 mg/ml, respectively.Another study revealed that the MIC and MBC of the aqueous extract of moringa leaf for Staphylococcus aureus were both 25 mg/ml, and the MIC and MBC for Escherichia coli were 12.5 and 50 mg/ml, respectively (Unegbu et al., 2020).The MIC and MBC are used to determine the presence of antibacterial activity; therefore, the current results revealed that the aqueous ME exhibits antibacterial activity against Vibrio.Mogana et al. (2020) demonstrated that an MBC:MIC ratio of <4 is bactericidal and an MBC:MIC ratio of >4 is bacteriostatic.Because the MIC and MBC had the same values, the ME is considered bactericidal for the Vibrio bacteria.The antimicrobial activity of the aqueous ME can be attributed to the presence of secondary metabolites, including phenols, flavonoids, steroids, glycosides, tannins, saponins, alkaloids, anthraquinones, and terpenoids (Unegbu et al., 2020).However, high MIC and MBC values for L. acidophilus indicate that high ME concentrations are bacteriostatic and not effective against L. acidophilus.
A low or negative prebiotic score means that the growth of the tested strain is lower on a specific prebiotic than on glucose or its growth is lower than that of the reference enteric bacteria (Huebner et al., 2007).A lower ME prebiotic score for V. alginolyticus compared with for V. harveyi and V. parahaemolyticus revealed that the ME inhibits the growth of V. harveyi and V. parahaemolyticus more than that of V. alginolyticus.However, a positive prebiotic score demonstrated that L. acidophilus grows more after metabolizing the ME than after metabolizing glucose.Moreover, L. acidophilus grows more effectively than the three Vibrio bacteria that metabolized the ME.
Administering probiotics and prebiotics in Penaeus vannamei affects the composition of the gut microbiota (Munaeni et al., 2020;Patil et al., 2021).Diet supplementation with ME and L. acidophilus in the ME2.5 + P and ME5.0 + P groups increased the presumptive lactic acid bacterial counts.The higher lactic acid bacterial count in the ME2.5 + P group indicated that the ME extract supports the growth of lactic acid bacteria in the gut.Prabawati et al. (2022) revealed that the symbiotic relationship between oyster mushrooms (Pleurotus ostreatus (Jacq.)P. Kumm., 1871) and L. plantarum significantly increased the number of lactic acid bacteria in the intestine; this finding is similar to that of our study.
The number of Vibrio-like bacteria did not significantly differ among the groups after feeding for 7 days.Imaizumi et al. (2021) reported that the probiotic diet did not alter the Vibrio bacterial count in the stomach and midgut after Penaeus vannamei were fed for 1 and 2 weeks.However, a study demonstrated that after 60 days of feeding, the Vibrio bacterial abundance was lower among shrimp fed with a diet supplemented with ME and L. acidophilus than among shrimp fed with a diet supplemented with ME alone (Abidin et al., 2022a).Another study indicated that the symbiotic effect of β-glucan with Bacillus subtilis (Ehrenberg, 1835) Cohn, 1872 reduced the number of Vibrio spp.and increased the number of lactic acid bacteria in whiteleg shrimp after 90 days of feeding (Boonanuntanasarn et al., 2016).However, the presence of Vibrio bacteria does not always affect shrimp, because some Vibrio bacteria are endogenous in the P. vannamei intestine (Zoqratt et al., 2018).
Incorporating moringa in diets increases the growth performance of some aquatic animals, such as the whiteleg shrimp, Penaeus vannamei (cf.Akbary et al., 2021;Abidin et al., 2022b), the giant freshwater prawn (Macrobranchium rosenbergii (De Man, 1879)) (cf.Kaleo et al., 2019), and nile tilapia (Oreochromis niloticus (Linnaeus, 1758)) (cf.Richter et al., 2003;Astuti et al., 2007;Hamed & Sayed, 2019;Gawad et al., 2020).Moreover, also inclusion of L. acidophilus in diets increased the growth performance of P. vannamei (cf.Wang & Gu, 2010;Liu et al., 2016;Zeng, 2019).The current study revealed that diet supplementation with ME and L. acidophilus, either individually or combined, increased the growth performance of P. vannamei.The ME2.5 + P group demonstrated higher growth performance than did the ME2.5 group, which indicated a symbiotic relationship between ME and L. acidophilus.The interaction of herbs and probiotics occurs when bacteria digests herbs into small molecules, which can then be easily absorbed by the host cell (An et al., 2019).Furthermore, the symbiotic effect between prebiotics and probiotics increases the villi height of the intestine of P. vannamei and the digestive enzyme activity (Yu et al., 2009;Boonanuntanasarn et al., 2016).Consequently, nutrient absorption and growth performance were improved in shrimp that were fed a diet containing ME and L. acidophilus.
In conclusion, the aqueous ME stimulates the growth of L. acidophilus and demonstrates antibacterial activity against Vibrio alginolyticus, V. parahaemolyticus, and V. harveyi.The ME demonstrates prebiotic activity and exerts a symbiotic effect that increases the growth performance of Penaeus vannamei when the ME is supplemented in diets at a concentration of 2.5 g/kg.

TABLE I
Ingredients of the experimental diets used in this study