Effects of Bacillus subtilis and Lactobacillus plantarum probiotics on the Litopenaeus vannamei growth performance, hemolymph factors, and physicochemical parameters

doi:10.21203/rs.3.rs-2341235/v1

Effects of two probiotics, Bacillus subtilis (ATCC 6633) and Lactobacillus plantarum (RITCC 1273) on the Litopenaeus vannamei postlarvae were investigated. Bacillus (B₁)10⁶ and (B₂) 3×10⁶ CFU.L^− 1 and Lactobacillus (L₁) 10⁵ and (L₂) 3×10⁵ CFU.L^− 1) were added to water in 5 treatments of different combinations of B₁, B₂, L₁, and L₂ to determine water quality, Hemolymph indices, salinity stress, growth rate, and productive parameters. after that, shrimps were transferred to seafood processing and quality control lab to evaluate shelf-life and quality change during freezing preservation at -18℃ for 90 days. Nutritional values, pH, Thiobarbituric acid reactive substances (TBARS), Total volatile nitrogen determination (TVB-N), Water holding capacity (WHC), drip loss, and texture profile were evaluated. According to the results, water quality remained almost the same during the aquaculture phase of the experiment and no significant changes were observed in the pH, ammonia nitrogen, nitrate-nitrogen, and nitrite-nitrogen of water. Growth performance tests indicated that T₁ (10⁵ L*3×10⁶ B CFU.L^− 1) had the highest weight gain (WG), survival rate (SR), specific growth rate (SGR), and relative feed conversion efficacy (RFC). T₄ (3×10⁵ L*10⁶ B CFU.L^− 1) indicated lowest triglyceride (274.51 ± 17 mg/dL) and highest cholesterol level (3581.61 ± 1222 mg/dL) significantly. Storage of shrimps demonstrated that the best performance belongs to T4 which had the highest WHC (31.9 ± 7.8%) and lowest drip loss (4.7 ± 0.4%) among treatments at day 60. Overall, our results showed interaction of B. subtilis and L. Plantarum significantly improves shrimp's growth rate, and helps to improve some of the chemical characteristics during storage at -18℃.

Aquaculture

Freezing Preservation

Nutritional Values

Shelf-life

Shrimp Postlarvae

Triglyceride

In the 20th century, antibiotics were widely used for controlling diseases and economic loss. Moreover, the disadvantages of antibiotics lead to selective pressure on bacterial resistance and invasive horizontal transferring of resistance genes among bacteria communities in reservoirs which spread these genes through the aquatic environments (Kumar et al. 2016), and residual antibiotics in seafood and aquatic products affect human health negatively (Qi et al. 2009). Furthermore, common antibiotics used in aquaculture kill or inhibit gut microflora natural ecosystem, affecting seafood quality and the immune system of aquatic animals (Banerjee and Ray 2017). In this regard, European Union 2003 prohibits the procedure of antibiotics in aquaculture practices (Ringø 2020). Infectious disease in aquaculture systems causes problems concerning aquatic farming in the world in high stocking densities and stressful conditions (Moradi et al. 2022; Kuebutornye et al. 2019). For this reason, a significant shift is necessary to use alternatives such as probiotics because they are safe and environmental-friendly agents (Hossain et al. 2017).

Terrestrial probiotics are different from aquaculture probiotics recently introduced in the aquaculture industry. Due to the constant interaction of aquatic species with the outside ecosystem, there is no specific difference between a microbial community in and out of aquatic hosts (Fečkaninová et al. 2017). The aquatics probiotic definition introduces microorganisms that can survive in water and has beneficial effects on aquatics by improving responses to disease or promoting growth (Javanmardi et al. 2020). To improvement functional probiotics, full-scale experimentations, and better monitoring tools are required to make an evaluation (Toledo et al. 2019).

Probiotics have been applied in aquaculture since the 1980s. Moreover, new attention has been concentrated on aquaculture practices (Van Doan et al. 2020). The potential mechanism is unknown how probiotics prevent damage to pathogens though it might refer to opposition for nutrients and adhesion sites, inhibitory substances production, and even immunological effects (Puvanendran et al. 2021). For instance, bacterial infections such as Streptococcus have affected Tilapia production, and probiotics are sustainable alternatives to integrate prevention. Some probiotics have been discovered as advantageous additives such as Bacillus species enhance fish growth performance and immune system. Bacillus cereus NY5 and Alcaligenes faecalis Y311 have been used as probiotic treatment in Tilapia culture, and a reduction of phosphorus concentration was observed that affected beneficial microbiota and the mucosal immune enzyme in Nile Tilapia (Wang et al. 2020). In rainbow trout culture, Yersinia ruckeri was detected several years ago as an ordinary bacterial infection. Use of bacteria isolated from the intestine of the host as probiotic improve the productive parameters of rainbow trout (Medina et al. 2020).

The pacific white shrimp (Litopenaeus vannamei) or king prawn is a priceless species for aquaculture indusrty since this species represents 70% of the entire shrimp population (Jiao et al. 2020). White shrimp naturally distributed along the south and central coast of America, and due to the importance of industrial marine species L. vannamei, probiotics such as Bacillus subtilis have indicated promising results in prohibiting Vibrio pathogens and increasing growth values (Interaminense et al. 2018; Yeh et al. 2009). The Bacillus is a Gram-positive, non-pathogenic, and spore-forming bacteria found in air, water, and soil. Also, Bacillus is in the intestine flora of shrimp tacts as a probiotic associated to improve immunity and prohibit the colonization of pathogens (Chien et al. 2020). Besides, Bacillus species are found in the digestive tract of shrimp and produce a wide range of substances such as enzymes, antimicrobial peptides, and spore coats remaining in an inactive state for a long time (Kewcharoen and Srisapoome 2019). Among different probiotic species used in shrimp culture, Lactobacillus plantarum widely applied to defeat acute low salinity and to enhance the growth of L. vannamei (Rezaee Tavabe et al. 2020; Zheng et al. 2020).

Current research aims to monitor the interaction of two types of Lactic Acid Bacteria (LAB), Bacillus subtilis (ATCC 6633) and lactobacillus plantarum (RITCC 1273) as a probiotic additive in the water of L. vannamei culture and to identify the effectiveness of probiotics on shrimp growth performance, hemolymph factors and also, physicochemical characteristics in storage for 60 days at -18 ℃. The effects of probiotics depend on different related factors such as husbandry conditions, procedures, and the concentration of bacteria, strain, and shrimp species. So, results might be helpful preventing economic losses and the advanced performance of aquaculture and the food industry. To western Pacific shrimp cultivation in the world and the expansion of the use of probiotics, this study was conducted to determine the combined effect of the mentioned strains on the growth factors and carcass quality of western Pacific shrimp.

2.1. Shrimps culture

Post larva of Litopenaeus vannamei (Number = 600; Weight = 0.5 to 1.5 g) was obtained from the Iranian Shrimp Research Institute located in Bandar Bushehr, Iran, and they were kept under the cultural condition in the laboratory of the Persian Gulf Research Institute of Marine animals in Bandar Bushehr for 2 weeks before experimentation in 4 m³ reservoir tank. The water source was provided from the Persian Gulf directly by a pumping system that was kept 24 hours to sediment. Chlorination and UV filtration for sterilization were applied before utilization of the seawater source and kept in different tanks for dichlorination. 20 post larvae shrimp for each treatment were chosen randomly and transferred to square 300 L tanks for further experiments.

2.2 Probiotic preparation

The commercial probiotics were purchased from Tak Gene co. (Bandar Bushehr, Iran) for Bacillus subtilis in the dose of 2.5 × 10¹¹ CFU/g and from Pardis Roshd Mehregan Bioproducts Co. (Iran) for Lactobacillus plantarum in the dose of 2 × 10¹¹ CFU/g and they were applied in two doses in experimental design.

2.3. Treatment procedures

Commercial probiotics in two doses of B. subtilis 10⁶ (B₁) and 3 × 10⁶ (B₂) CFU/gr and L. Plantarum 10⁵ (L₁) and 3 × 10⁵ (L₂) CFU/gr were used. The tanks of the experiment were designed into five groups: T₁ = L₁ × B₂, T₂ = L₁ × B₁, T₃ = L₂ × B₂, T₄ = L₂ × B_1, and a Control sample with three replications. After the adaptation period, 300 post-larvae were divided equally into 15 of 0.3 m³ tanks randomly with electric aeration grills placed at the bottom to supply oxygen and water circulation. The bacterial solutions were added two times per week after water exchange during of experiment (60 days).

2.4. Feeding

The larvae were fed with a commercial pelleted diet from Havorrash Co. (Iran) three times daily and the amount of feed was calculated by the mean weight of shrimp (10–15% of weight). The composition of feed was observed according to the Association of official analytical chemist guidelines (AOAG, 1997) and is indicated in Table 1. The water in tanks was changed 2 times per week and probiotics were added after every water exchange.

Table 1

Percentage of composition in the pelleted diet of shrimps during the experiment.
Chemical analyses	Initial			growth				ending
size	4001	4002	4003		4004	4005			4006
Minimum Protein (%)	40			29			28	36
Minimum fat (%)	8			7			7	6
Maximum Ash (%)	11			12			13	13
Minimum Fiber (%)	2			3			3	3
Maximum moisture (%)	10			10			10	10

2.5. Water quality examination

The temperature, pH, ammonia nitrogen, nitrate-nitrogen, and nitrite-nitrogen were measured daily (pH and temperature) and weekly (Nitrogen compounds). Waterproof (HI98127 Checker, Hanna Instruments, USA) and the AQUAPA kit of Payam Abzian Arvand co. (Iran) were used for monitoring pH and temperature, and chemical compounds level. According to this purpose, water samples were collected (0.1 L) and then centrifuged.

2.6. Hemolymph sampling

After 60 days when shrimps gained weight, hemolymph sampling (100 µL) from the ventral-sinus cavity of Litopenaeus vannamei was collected by a 26-gauge needle, and heparin was used to prohibit coagulation (6 samples for each treatment). Then, samples were centrifuged under the sterile condition at 4 ℃, 1000 g for 15 min, and supernatants were collected and kept at -20 ℃ for further analyses. Total proteins, albumin, triglycerides, and cholesterol were examined according to the guidelines of Pars Azmun Co. kits (Iran).

2.7. Salinity stress evaluation

End of the examination period, tanks were prepared in 70 g/l salinity and sea salt was used. Six shrimp from each sample were randomly selected and transported to high salinity tanks. The time of lethargy and mortality were recorded.

2.8. Growth performance parameters

At the end of culture duration, all shrimps were harvested and weighted to examine average weight and parameters such as survival rate (SR), weight gain (WG), specific growth rate (SGR), feed conversion rate (FCR), and relative feed conversion efficacy (RFC) according to Eq. (1, 2, 3, 4, and 5) (Chowdhury and Roy 2020):

WG (g) = W ₁ -W ₂ (1)

FCR (%) = feed consumed/ WG (2)

SGR (%) = [{Ln (W ₁ ) – Ln (W ₂ )}/ 60 (days)] × 100 (3)

SR (%) = (final survived shrimps/ stocked shrimps) × 100 (4)

RFC (%) = [{W ₂ (g) – W₁ (g)}/ W₁ (g)] × 100 (5)

W₁: initial weight, W₂: final weight.

2.9. Chemical and physical analyses

Harvested shrimps were kept on ice and transported to a seafood processing laboratory at the University of Tehran to analyze the chemical and biochemical characteristics of shrimps for 60 days and all tests were applied on days 1, 15, 30, 45, and 60.

2.10. Nutritional values of shrimps

Shrimps were selected to evaluate protein, moisture, and fat content. According to this purpose, samples were dried in the oven at 60 ℃ for 8 h to evaluate moisture content, and then the content of fat and proteins were examined. In short, crude protein content was determined according to the Kjeldahl method (AOAC, 1995) and fat content was analyzed using the gravimetrical method by using petroleum ether for extraction of fat (ASU L 13.05-3).

2.11. pH

The pH of treatments was determined as the previously described methodology (Alirezalu et al. 2020). The test was applied using a pH meter (Microprocessor pH meter, HANNA instrument, USA) and briefly, 5.0 g of samples were homogenized with 45 ml of distilled water then, pH was read at 20 ℃.

2.12. Thiobarbuturic acid reactive substances (TBARS)

TBARS (mg of malondialdehyde (MDA)/kg of shrimps) test was carried out to observe lipid oxidation during the storage. The method that has been used previously is on fish fillets with slight modification (Chatzikyriakidou and Katsanidis 2012). In brief, 3.0 g of minced samples were mixed with 30 ml trichloroacetic acid (0.45 M) solution with 0.1 EDTA and were homogenized by using a homogenizer (T25D, IKA, Germany) for 1–3 min. the homogenized samples were transferred to a centrifuge at 8000 g for 10 min (4 ℃), then the supernatant was filtered using Whatman n°3 filter paper. 2 ml of thiobarbituric acid (0.28% w/v) was added to 2 ml of supernatant test tubes in the ratio of 1:1, then they were heated using a boiling water bath for 45 min. After a while, the color of the samples was developed. The absorbance of solutions in different samples was obtained after letting them cool down at room temperature at 532 nm using an Ultraviolet-Visible Spectrometer (Perkin Elmer, USA). The result was explained as mg malondialdehyde (MDA/KG) and it was calculated with a 5.1 factor.

2.13. Total volatile nitrogen determination (TVB-N)

TVB-N content was carried out using a Micro-diffusion assay (Rawdkuen et al. 2010). Briefly, 2 g of minced samples was added to 8 mL of TCA 4% (w/v) and homogenized at 10000 rpm for 3 min. Then, samples were centrifuged at 3000 g for 10 min at 20 ℃. The supernatants were collected and mixed with K₂CO₃ and squeezed into the outer ring of the Conway micro-diffusion apparatus and the inner ring was poured with boric acid 1%, bromocresol green, and methyl red indicator then, kept at 37 ℃ for 1 h. the inner ring was titrated with HCl 0.02 N to change color from green to pink and volatile nitrogen was calculated by the Eq. (6) below:

Total Volatile Nitrogen (mg N/100 minced sample) =\(\frac{14\times N \times (A-B)\times V\times 100}{M}\)(6)

Where N is acid HCl normality, A and B are HCl titration in samples and blank respectively, the volume of sample and M is sample mass (g).

2.14 Water holding capacity changes

Water Holding Capacity (WHC) will be examined by the previously described method (Sánchez-Valencia et al. 2014). 3 g of minced samples were weighed into the culture tube and placed both filter cloth (2 Whatman paper n°1), and tube into the syringe barrel. For the final step, the syringe will be placed into the 50 mL conical centrifuge tube to centrifuge at 3000 g for 15 min at 4 ℃. WHC of each sample was measured at days 0, 15, 30, 45, and 60 triplicate, and WHC was expressed as % of the water that was retained after centrifuge.

2.15 Determination of drip loss

The drop loss of treatments was determined using frizzed whole shrimps under − 18 ℃ for 2 weeks. In brief, after thawing the sample at 4 ℃ till the temperature of the central point of the shrimp reached 0 ℃, tissue was used to absorb water droplets of shrimp samples. Samples were weighed after and before drip loss. Whole shrimps should be prepared in a suitable plastic package to avoid water evaporation from the surface of the product (Yuan et al. 2020).

2.16 Texture profiling

Texture profile analyses (TPA) of the shrimps were carried out using the texture analyzer (CT310K, BROOKFIELD, USA) with the 4 mm diameter cylinder (TA/44) probe. The trigger load was 0.7 N and hardness and springiness parameters were determined at room temperature. The settings were a 2-cycle test including a pre-test speed of 2.00 mm/s, a test speed of 0.50 mm/s, and a return speed of 0.5 mm/s. Textural changes were observed through 60 days of storage. The Texture Expert software (TexturePro CT V1.8 Build 31) was used for gathering and processing the data (Ozaki et al. 2020).

2.17 Statistical analyses

Tests and analyses were carried out in triplicate and reports were indicated as mean along with standard deviation. The SPSS for Windows (SPSS Inc., 22.0, Chicago, IL, USA) was used to determine significant differences between the mean of each variable. Two-way analyses of variance (ANOA) and the Duncan test (p < 0.05) were applied.

3.1 Water quality evaluations

the temperature, and pH of water, ammonia, nitrate, and nitrite are almost the same in all treatments which means water quality measurements are not significantly (p > 0.05) different in all treatments/days due to regular water changes in tanks (twice per week) and probiotics were added after each change. Water temperature fluctuated between 24 ± 0.5 ℃ at the end of September to 18 ± 0.2 ℃ in early November during of culture. pH, ammonia, nitrate, and nitrite were measured 7.65 ± 0.016, 0.5 ± 0.67 mg/L, 18.5 ± 4.5 mg/L, and 2 ± 2.02 mg/L respectively.

1.1. Determination of growth and survival

The probiotics and their interactions had significant effects on the growth performance of shrimp in some treatments (p < 0.05) which is shown in Table 2. The mean final biomass (g) was the highest in T₁ (177.65 ± 18 g) which is significantly different from C (130.62 ± 29 g) and T₂ (124 ± 31 g) and also insignificant to T₃ (150.07 ± 9 g) and T₄ (157.92 ± 5 g). Also, B. subtilis and L. Plantarum had no significant effects on growth (p > 0.05). However, the interaction of B. subtilis and L. Plantarum was significantly recorded.

There was no significant difference among treatments in FCR (p < 0.05). Furthermore, the highest specific growth rate (SGR) (%) found in T₁ (3.52 ± 0.1%) was significantly higher than T₂ (2.97 ± 0.26%) and was insignificant to C, T_3, and T₄ and also, b. subtilis has no significant feed conversion rate among treatments (p = 0.158). The survival rate (SR) was different significantly among treatments. The highest percentage found in T₄ (91.67 ± 2%) was different from T₂, T_3, and C and also was insignificant to T₄ which means B. subtilis × L. Plantarum affects survival rate (p = 0.008).

The highest RFC was found in T₁ (726.29 ± 54.45%) which is significantly different from C (568.53 ± 160.89%) and T₂ (498.56 ± 94.40%) with applying different content of probiotics and there is no significant difference between T₃ and T₄ (p > 0.05). In this test, B. subtilis affected significantly all treatments (p = 0.035).

The result of mortality during the salinity challenge can be found in Table 3. As can be seen, treatments survived until 90 minutes and after that, the mortality started and at 150 minutes the differences among treatments are significantly different. The highest mortality rate belongs to C (4.3 ± 0.5%) and the lowest belongs to T₁ (1.3 ± 0.5). Also, Lactobacillus and Bacillus effects significantly (p < 0.05) the survival of shrimps in salinity separately.

Table 3

the number of shrimp mortality during 150 minutes of salinity challenge (70 g/L).
Treatments			Time (min)
	30	60	90	120	150
C	0 ± 0^a	0 ± 0^a	1.3 ± 0.5^a	1.3 ± 0.5^a	4.3 ± 0.5^d
T1	0 ± 0^a	0 ± 0^a	1 ± 0.5^a	1 ± 0^a	1.3 ± 0.5^a
T2	0 ± 0^a	0 ± 0^a	1.3 ± 0.5^a	1.3 ± 0.5^a	2.3 ± 0.5^bc
T3	0 ± 0^a	0 ± 0^a	1.3 ± 0.5^a	1.3 ± 0.5^a	2 ± 0^ab
T4	0 ± 0^a	0 ± 0^a	1.33 ± 0.5^a	1.3 ± 0.5^a	3 ± 0^c
				p-value
Bacillus	...	...	0.6	0.5	0.003
Lactobacillus	...	...	0.6	0.5	0.02
L×B	...	...	0.6	0.5	1
Values are presented as the means ± SEM of three replicates (n = 3). Values in the same row with different superscripts are significantly different (P < 0.05).

1.2. Hemolymph evaluations

Hemolymph factors were examined and given Table 3 shows the results. There were no differences (p > 0.05) in protein content of all treatments and control group hemolymph. However, the highest content of protein in hemolymphs of L. vannamei was found in T₁ (13.63 ± 5 g/dL).

Albumin levels in serum of hemolymphs showed a significant difference in the level of 0.05 between the control group and T₃ and there are no statistical differences between T₁, T_2, and T₄. The highest level of albumin belongs to C (3.55 ± 0.42 g/dL) and the lowest level is T₃ (2.60 ± 0.47 g/dL).

Cholesterol level indicates a significant difference between T₂ and other treatments which is the lowest amount of cholesterol among others (314.94 ± 51 mg/dL). Also, the simultaneous effects of Lactobacillus and Bacillus are significant (p = 0.009).

Different levels of Bacillus and Lactobacillus had significant effects on triglyceride levels among treatments which mean there are statistical differences between T₁, T_2, and T₄. T₄ is significantly different from other treatments. Lactobacillus and Bacillus had effects individually and simultaneously on triglyceride levels.

1.3. Quality evaluation of shrimp during preservation

1.3.1. Determination of the proximate composition of shrimp treatments

Determination of the proximate composition of shrimp treatment results is indicated in Table 5. There is a significant difference (p < 0.05) in Crude protein level of T₄ (86.05 ± 0.17%) and other treatments and probiotic treatments had statistical effects on the crude protein content as well as fat concentration. There is a significant difference between T₄ and T₃ in Total Fat % meaning that the highest amount of Total Fat belongs to T₄ (2.46 ± 0.07). Also, there is no meaningful difference in Fat % between C, T1, and T2. Moisture results show no significant difference among treatments.

1.3.2. Physio-chemical evaluation of stored shrimps over 60 days

Water holding capacity, pH, TVB-n, TBA, drip loss, and textural evaluations (springiness and hardness) were observed in specific intervals (0, 15, 30, 45, and 60 days of storage) at 4 ℃ to monitor quality changes for further usage. WHO was changed during 60 days as can be found in Fig. 1. WHC was increased in all treatments until day 15 except T₂ and then decreased at day 60 (p < 0.05). There is no significant difference between treatments in each interval except on day 60 which shows a difference between T₄ (31.9 ± 7.8%) and C (18.1 ± 4.8%).

pH data shows an increase over 60 days (Fig. 2.). Initial pH which is the same in all treatments increases steadily till reached the highest level of T2 (7.4 ± 0.03). Also in all intervals, T₂ and C showed significant differences among other means that T₂ always had the highest pH and the lowest level belonged to C.

Given Fig. 4. shows the TVBN increase from day 0 to day 60. Although TVBN increased over 60 days in all treatments, meaningful differences can not be recognized among treatments each day meaning that probiotics had no effects on the TVBN level. The lowest level of TVBN belongs to T₂ (12.1 ± 5.6) on day 0 and the highest comes to T₂ (26.1 ± 0.8) on day 60.

TBARS graph (Fig. 3) indicates that TBARS increased over 60 days of storage and there is a significant difference between treatments. For example, T₂ (0.02 ± 0.005 mgMal/100g), T₃ (0.01 ± 0.005 mgMal/100g) and C (0.05 ± 0.0 mgMal/100g) are different statistically at day 15.

There is no significant difference in drip loss among treatments for all sampling days (Fig. 5). Overall, drip loss had some fluctuations during storage days. Springiness shows an overall increase for all treatments as the lowest springiness belongs to first-day samples and fluctuations for hardness (Table 6).

Table 6

textural evaluation of stored shrimps over 60 days.
Treatments P-value
		C	T₁	T₂	T₃	T₄	Lactobacillus	Bacillus	L×B
Springiness (MM)	1	0.98 ± 0.01^a	1.11 ± 0.04^a	1.07 ± 0.08^a	1.07 ± 0.01^a	1.13 ± 0.03^a	0.9	0.9	0.07
	15	0.98 ± 0.01^f	1.02 ± 0.02^f	1.39 ± 0.01^f	1.18 ± 0.03^f	1.03 ± 0.01^f	0.2	0.2	0.02
	30	1.27 ± 0.01^j	1.29 ± 0.01^kl	1.26 ± 0.04^l	1.33 ± 0.04^l	1.28 ± 0.02^k	0.6	0.7	0.8
	45	1.07 ± 0^p	1.11 ± 0.02^qr	1.14 ± 0.01^r	1.22 ± 0.01^q	1.29 ± 0.01^o	0.1	0.005	0.5
	60	1.16 ± 0.03^t	0.91 ± 0.03^u	0.95 ± 0^t	1.06 ± 0.02^u	1.05 ± 0.01^t	0.06	0.01	0.4
Hardness (N)	1	0.97 ± 0.3^a	1.03 ± 0.3^a	0.86 ± 0.1^a	0.81 ± 0.3^a	0.95 ± 0.2^a	0.9	0.5	0.2
	15	1.07 ± 0.3^f	1.21 ± 0.1^f	1.19 ± 0.3^f	1.24 ± 0.3^f	1.11 ± 0.1^f	0.5	0.8	0.6
	30	1.04 ± 0.2^j	0.67 ± 0.1^j	0.79 ± 0.6^j	1.06 ± 0.3^j	1.03 ± 0.4^j	0.7	0.04	0.5
	45	1.04 ± 0.1^o	1.04 ± 0.1^o	0.95 ± 0.9^o	0.79 ± 0.7^o	0.94 ± 0.2^o	0.7	0.1	0.1
	60	1.04 ± 0.1^tu	1.06 ± 0.1^tu	0.87 ± 0.1^t	1.19 ± 0.2^u	1.05 ± 0.1^tu	0.04	0.06	0.6

2.1. Growth performance

The use of probiotics plays an important role nowadays due to the importance of eco-friendly aquaculture practices. For example, probiotics tend to increase water quality and enhancement of nutritional value in host species since it helps the digestion system and produces supplemental digestive enzymes, reduce disease, help to greater survival rate, and boost the immune system (Gullian et al. 2004). Also, different probiotics were recommended for fish and shellfish such as Bacillus and Lactobacillus (Zhou et al. 2009). T₁ showed the highest WG, RFC, SGR, and SR which had a significant difference from other treatments means that L₁ × B₂ affects the growth of shrimps. Enhancement of the SR in T₁ is due to the increasing number of Bacillus probiotics in the digestive tract of shrimps causing the production of extracellular enzymes as well as acting as a symbiotic association to improve growth performance and provide necessary growth factors (Balcázar et al. 2007; Nimrat et al. n.d.).

According to the literature review, a high dose of Bacillus subtilis may increase WG% and SR%. Several studies focused on Bacillus in shrimp aquaculture that indicated improvement in farmed shrimp growth and survival percent because Bacillus enzymes break down nutrients such as proteins, carbohydrates, and lipids into smaller parts that help shrimps to digest efficiently (Liu et al. 2009). Also, bacillus subtilis is produce a wide range of extracellular antimicrobial peptides that avoid the growth of other microorganisms which reduces mortality (Interaminense et al. 2018). Other scientists' results show that lactobacillus Plantarum addition at 5.0 × 10⁶ CFU/ml enhances SR% and enzyme activity so, T₄ high SR% may result due to higher addition of L. Plantarum (3 × 10⁵) (Talpur et al. 2013). Also, the digestive system of the larval stage of shrimp is more active therefore, probiotics have better effects (Wang 2007).

2.2. Hemolymph parameters and salinity challenge

There is no significant difference in protein, cholesterol, and albumin concentration between C and other treated groups that is the same as in previous examinations (Gullian et al. 2004; Li et al. 2007) although, some differences are crystal clear. Cholesterol is one of the most noteworthy molecules in the cellular membrane, and it is critical for function and structure. Also, cholesterol plays an important role in steroid hormones which is important for growth and the life cycle (Pourmozaffar et al. 2019). Due to less concentration of cholesterol in T2, the SGR and RFC % were low because Litopenaeus vannamei does not have the ability of de novo sterol synthesis from acetate to a deficiency of dietary cholesterol leads to reduce growth performance (Morris et al. 2011).

As far as shrimps were fed with the same pellet, T2 treatments cannot metabolize cholesterol, or the intestine microbial balance might not let them absorb cholesterol from feed. It is accepted that lipids such as triglycerides play a key role in proper growth and maturation (Yepiz-Plascencia et al. 2000). The ability of triglyceride uptake might increase in T₁ leads to growth better and results show an increase in SGR, WG, and SR%. Probiotics provide beta-glucan and nucleotides that can help the immune system in fish (Faramarzi et al. 2012), and in current research, the probiotics have significant effects separately on the survival of shrimps in salinity challenges.

2.3. Shelf-life quality and nutritional value evaluations

The highest cholesterol level was found in T4 (p < 0.05), moreover, higher protein and fat content belong to T4 which also has a higher survival rate. Shrimp proteins mainly comprise myofibrillar and sarcoplasmic proteins. In a literature review, a lower percentage of protein content has been recorded varied from 72.0 ± 1.5 to 74.1 ± 0.1% in Litopenaeus vannamei (Jatobá et al. 2014). According to Tsai et al. (2019) findings, there are no differences between the Control group and the treatment fed with Bacillus subtilis E20 (109 CFU/kg) (Tsai et al. 2019) in protein, fat, and moisture content.

2.4. Quality evaluations

Many seafood properties of appearance and textural depend on WHC. In addition, changes in protein structure decrease WHC (Pan et al. 2019). WHC of T4 at the end of storage duration (31.9 ± 7.8%) is the highest which confirms the result of the highest protein content of T4. Similar observations had been carried out for Litopenaeus vannamei during ice storage which is explained initial increase by the fact that protein in muscle oxidizes before the lipids lead to move water from myofibrils into extracellular spaces resulted enhances of WHC (Mehta et al. 2021). pH changes during 60 days of storage are probably due to the formation of TVB-N developed from microbial activity and leads to the formation of non-protein formation compounds (Gonçalves and Lira Santos 2019). Also, probiotics had no significant effects on groups (p > 0.05). The TVB increase is consequential as an indicator of chemical changes in shrimps since it is associated with microorganisms and endogenous enzyme activity. The acceptable threshold of TVB-N is < 25 mg%, and higher than this amount is considered spoiled and not edible (Kazemzadeh et al. 2022; Na et al. 2018). On the last day of the experiment, treatments exceeded the TVB-N value > 25 mg%. However, the low temperature of preservation slowed down the microbial activity and effects of enzymes, so the frozen-stored shrimps could not show differences among treatments.

Lipid oxidation and production of residuals from polyunsaturated fatty acids which are highly reactive and further reaction cause produce secondary oxidation species that lead to off-flavors, loss of nutrients, and shorter shelf-life (Zhang et al. 2015). There are no probiotic effects on TBA and no significant changes among treatments were detected. The drip loss value indicates that myofibrillar proteins denatured and the water-binding capacity of proteins decreased (Logan et al. 2019). T₄ treatment which had the highest protein content also has lower drip loss%. The texture of the muscle comprises mainly myofibrillar proteins and connective tissue effecting by storage conditions and temperature (Panini et al. 2017). Springiness and harness fluctuated during storage time and the effects of probiotics were not significant.

Overall, it was evaluated that the Bacillus subtilis (ATCC 6633) and lactobacillus Plantarum (RITCC 1273) as probiotic candidates, significantly improve shrimps’ growth performance, and also chemical evaluation of hemolymph and composition indicated that probiotics effects shrimps. Synergistic effects of lactobacillus and bacillus can be observed over storage evaluations. Finally, the effects of probiotics are more obvious in the aquaculture phase and can improve shrimps’ growth performance.

Author contribution:

Seyed Mehrdad Hasani Azhdari: Investigation and statistical Analysis.

Kamran Rezaei Tavabe: Funding acquisition, conceptualization, and review.

Shirin Kazemzadeh Pournaki: Writing, and research performances.

Sina Javanmardi: Review and editing.

All authors have participated in (a) conception and design or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version.

Funding: The current study was funded by the University of Tehran under grant number 26713.

Data availability: The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary material.

Code availability: Not applicable.

Ethics statement: The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to, and the appropriate ethical review committee approval has been received. The proposal and protocols of the present work was reviewed and approved by this scientific committee. The trial protocol was approved by the Ethics Committee for the Animal Research, University of Tehran; none of the fish suffered starvation, trauma, or electrical shock, and all the fish were completely anesthetized before tissue sampling.

Consent to participate: All names in author list have been involved in various stages of experimentation or writing.

Consent for publication: All authors agree with submission of the paper for publication in the journal of Aquaculture International.

Competing interest: The authors declare no competing interests.

Acknowledgements:

The authors wish to thank the Fisheries Department of Natural Resources Faculty of the University of Tehran for providing experimental facilities.

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Tables 2, 4 and 5 are available in the Supplementary Files section.

No competing interests reported.

Effects of Bacillus subtilis and Lactobacillus plantarum probiotics on the Litopenaeus vannamei growth performance, hemolymph factors, and physicochemical parameters

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