Bacterial protein meal digestibility, digestive enzyme activity and kinetics in barramundi (Lates calcarifer) and tilapia (Oreochromis mossambicus) juveniles

doi:10.21203/rs.3.rs-4811184/v1

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Bacterial protein meal digestibility, digestive enzyme activity and kinetics in barramundi (Lates calcarifer) and tilapia (Oreochromis mossambicus) juveniles

https://doi.org/10.21203/rs.3.rs-4811184/v1

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Single-cell protein meals, particularly bacterial protein meals (BPMs), show promise as alternative protein sources for fish diets. However, their digestibility in non-salmonid species remains underexplored. This study evaluated the apparent digestibility coefficients (ADCs) of a BPM derived from brewery by-products when fed to barramundi (Lates calcarifer) and tilapia (Oreochromis mossambicus). Two cohorts of 150 barramundi (48.9 ± 4.2 g, 95 dph) and 150 tilapia (48.7 ± 11.4 g, 219 dph) were raised in recirculated systems with brackish water (28°C, 5 ppt salinity) over two trials. Fish performance, ADCs for 42 fatty acids and 17 amino acids, and gut enzyme activity, including the Michaelis–Menten constant (Km) for trypsin, chymotrypsin, and leucine aminopeptidase, were assessed. Both species exhibited high thermal-unit growth coefficients (1.7–1.9) and low feed-conversion ratios (1.1) on control diets. Barramundi had a slightly but significantly higher diet ADC for protein (95.8 ± 0.5%) compared to tilapia (94.8 ± 0.6%) (P < 0.05), consistent across most essential amino acids except lysine. Conversely, barramundi showed lower digestibility for PUFA (92.0%) and omega-6 (85.7%) than tilapia. The ADCs of BPM for dry matter and protein were higher in barramundi (66.3 ± 5.8% and 87.5 ± 0.9%) than in tilapia (54.1 ± 5.1% and 71.8 ± 5.3%). Tilapia exhibited higher gut enzyme activities than barramundi, and these activities were unaffected by BPM incorporation (P > 0.05), indicating no presence of digestive inhibitors in the tested BPM. The affinity of digestive trypsin in both species (Km = 11.8–15.1) was 3 to 30 times higher than that measured in other fish species. High affinities for chymotrypsin, and leucine aminopeptidase were observed in both tilapia (240.0 ± 58.6; 132.2 ± 3.2, respectively) and barramundi (425.7 ± 50.7; 70.0 ± 2.2, respectively) and significant interaction suggest that the relationship between diet and enzyme kinetics varies between species. This study demonstrates that both barramundi and tilapia can effectively digest the tested BPM, with barramundi showing superior efficiency. It underscores the importance of tailoring aquafeed ingredients to specific species based on nutrient digestibilities and enzyme kinetics.

Biological sciences/Chemical biology/Enzymes

Biological sciences/Biochemistry/Proteases

Barramundi

mossambicus

single-cell protein

digestibility

aquaculture

In recent decades, the limited availability of wild fish stocks, coupled with the growth of aquaculture, has led to the scarcity and escalating cost of fish meal, a primary protein source traditionally used in aquaculture feeds. In response, there have been efforts to identify and thoroughly evaluate alternative ingredients that can offer formulators the flexibility and adaptability required to navigate the dynamic landscape of aquatic feed production ¹. Among these alternatives, single-cell protein (SCP) meals have emerged as promising novel protein sources capable of partially or entirely substituting the traditional plant-based and animal protein sources used in fish diets ². SCPs are typically dried cellular masses produced from algae, yeast, bacteria, and fungi. They have gained increasing attention as protein-rich supplements or ingredients for both human and animal diets ³. A type of SCP, bacterial protein meal (BPM) shows particular promise as an ingredient in aquafeed formulations. However, while the digestibility of BPM has been estimated for salmonids ⁴, no digestibility estimates exist for warmwater species such as barramundi and tilapia.

This investigation focused on a commercially available BPM composed of dried and autolyzed bacteria grown on brewery by-products (China). Barramundi (Lates calcarifer) and tilapia (Oreochromis mossambicus) are the two predominant farmed species in Singapore. Barramundi, a carnivorous diadromous fish requires protein levels ranging from 45–55% depending on their size ⁵. O. mossambicus tilapia, commonly known as saline tilapia in Singapore, is an opportunistic omnivore ⁶ with poorly understood protein requirements. ⁷ reported a 40% protein requirement, higher than that of other tilapia species. Domestication and genetic selection may have altered these needs, potentially explaining the observed optimum protein levels of 42–45% (unpublished data).

As proteases involved in fish digestion play pivotal roles in breaking down available food components ⁸ and are major contributing factors to the conversion of feed to growth via protein turnover ⁹, this study also explored the impact of BPM use on key proteases crucial for protein digestion in fish (Mai et al., 2022): trypsin, chymotrypsin, and aminopeptidase. This study represents one of the first attempts to characterize BPM digestibility in both O. mossambicus tilapia and barramundi and to provide a characterization of digestive enzyme activity and kinetics in these species. This research aimed to understand the digestion of BPM by two farmed fish species, with the goal of optimizing its use as an alternative ingredient in aquafeeds.

Ethical statement

All fish handling procedures were conducted in accordance with relevant guidelines on the care and use of animals for scientific purposes developed by the Singapore Institutional Animal Care and Use Committee (approval number: TLL(F)-23-001).

Fish and system

In two separate trials, two cohorts consisting of 150 barramundi (48.9 ± 4.2 g, 95 days post-hatching [dph]) and 150 tilapia (48.7 ± 11.4 g, 219 dph) were fed one of two experimental diets in triplicate tanks for 23 and 29 days, respectively. The barramundi cohort originated from the Barramundi Group hatchery, while the tilapia cohort came from a 6th generation of the Temasek Life Sciences Laboratory (TLL) breeding program that was introduced to Singapore from South Africa (University of Stellenbosch) in 2009. Fish were grown in nine round conical-bottom 300-L tanks, with 25 fish per tank, and subjected to a natural 12:12-hour photoperiod. The tanks were supplied with brackish water (5 ppt salinity) by a recirculated aquaculture system (RAS). The RAS included UV treatment and sand and biological filtration steps and supplied water to each tank at a flow rate of 12 L per minute (resulting in 250% tank renewal per hour). Parameters such as dissolved oxygen, salinity, temperature, pH, nitrate, nitrite, and total ammonia in the system water were measured daily. After 14 days of acclimatization in the system, the fish were individually weighed (± 0.1 g) following light sedation with MS-222 (100–200 mg L^− 1), and the same weighing procedure was repeated on the last day of the trial. On the penultimate day of each trial, two fish were sampled from each tank, euthanized in MS-222 (500 mg L^− 1) 1 hour after feeding, and their whole digestive tracts were collected and stored at − 80°C for proteomic analysis.

Diet production

The BPM was a commercial product (China) made of a dried and autolyzed mixture of bacteria grown on brewery by-products. A control diet (CTL) was formulated with Wittaya software (Wittaya Aqua International Inc., 2022) to be as compatible as possible with the nutritional requirements of barramundi ¹⁰ and saline tilapia in terms of digestible proteins, energy, and lipids. A CTL premix was prepared at the R&D feed mill of the Marine Aquaculture Centre (Singapore). Ingredients were hammer milled (Mikro AP-1SH, Hosokawa Micron, USA) and sieved to a particle size of 500 µm using a vibratory sifter. Subsequently, the ingredients were thoroughly dry-mixed by utilizing a powder mixer (KSE-PM100, Kong Shiang Engineering, Singapore), and yttrium oxide (Y₂O₃) was added as an indigestible marker. BPM diets were prepared by incorporating 30% of BPM into the CTL premix (Table 1). In a 60-L mixer (HL-600, Hobart, Pinkenba, Australia), premixes were mixed with fish oil and screw-pressed through a 3.175-mm diameter die using a meat mincer (HL-400, Hobart, Australia). The diets were subsequently dried at 65°C in an oven (Memmert VO200) for 24 hours, bagged, and stored at 4°C until use in the trials. Tested ingredients and diets were subjected to proximate composition analysis (Table 2).

Table 1. Formulation (g kg^-1) of experimental diets for both tilapia and barramundi¹

	CTL	BPM
Fish meal²	259.5	181.6
BPM³		300.0
Soybean meal	381.3	266.9
SPC⁴	187.2	131.0
Soy lecithin	30.0	21.0
Rice flour	89.8	62.9
Sardine oil⁵	43.8	30.7
Vitamin premix⁶	5.0	3.5
Mineral premix⁶	2.5	1.7
Yttrium oxide⁷	1.0	0.7

¹ CTL: Control diet; BPM: Bacterial protein meal

² FF-Classic, Skagen, Denmark, 70% crude protein

³ Bacterial protein meal made of a mix of autolyzed bacteria grown on brewery by-products (China)

⁴ Soy protein concentrate, X-Soy 200 (C.J. Selecta, Brazil), 60% crude protein

⁵ Sardine oil contained no antioxidants

⁶ ANA Fish Vitamin Premix-199 and ANA Fish Mineral Premix-199 (Zagro, Singapore)

⁷ Regent Chemicals Pte. Ltd., Singapore

Feeding and waste collection

Operators manually distributed the experimental diets (Table 1) at 11:00 AM five days a week at a feeding rate of 1.8% of body weight for barramundi and 2.5% of body weight for tilapia. Feed amounts were progressively adjusted based on the known thermal-unit growth coefficient (TGC) for each species and refined further based on weight gained during the acclimatization period. The uneaten pellets were removed and quantified after 30 min of feeding. During the last 6 days of the acclimatization period, fish were fed the control diet to minimize the influence of the commercial diet. As per the recommendation of Blyth et al. (2015), fecal collection started on day 4. Feces within the conical-shaped-bottom tanks accumulated in a customized collector at the base of each tank, which was collected daily over 13 days. The fecal samples were centrifuged at 3,900 × g for 10 minutes at 4°C, with the supernatant being discarded, and the pellets were frozen at − 20°C before being analyzed for their chemical content.

Chemical analysis

Chemical analyses of diets and feces were conducted by an accredited analytical service laboratory (Eurofins, Singapore) following the Association of Official Analytical Chemists methods ¹². Dry Matter (DM) content was calculated by gravimetric analysis (AOAC 925.09) following oven drying at 100°C for 5 hours and desiccator cooling. Crude lipid (CL) content was estimated by acid hydrolysis (AOAC 922.06). Ash content was determined gravimetrically following the loss of mass after combustion of the sample at 550°C (AOAC 923.03). Total nitrogen (N) was estimated after pyrolysis and combustion (AOAC 968.06), and crude protein (CP) levels were based on N × 6.25. Crude fiber (fiber) was estimated after the digestion of the sample with sulfuric acid and sodium hydroxide (AOAC 962.09). Nitrogen-free extract (NFE) was calculated using the DM-CP-CL-fiber-ash formula, and gross energy (GE) was determined using the formula 23.6 × CP + 39 × CL + 17.6 × (fiber + NFE), as detailed by the National Research Council (1993). Fatty acids (FAs) in the samples were methylated to FA methyl-esters and quantitively measured by capillary gas chromatography (AOAC 996.06). The complete list of measured FAs used to compute omega-3 (n-3), omega-6 (n-6), omega-7 (n-7), omega-9 (n-9), saturated fatty acid (SFA), mono-unsaturated fatty acid (MFA), poly-unsaturated fatty acid (PUFA), and long-chain poly-unsaturated fatty acid (LC-PUFA) levels is provided in Table A1. Following digestion in hydrofluoric acid at 80°C, yttrium was quantified by a separate analytical service laboratory (Latech, Singapore) using inductively coupled plasma mass spectrometry. The amino acid composition of samples was determined by acid hydrolysis prior to separation using high-performance liquid chromatography, as explained by Schuster (1988).

Table 2. Composition (g kg^-1 DM unless otherwise indicated) of ingredient and experimental diets

	Ingredient	Tilapia trial		Barramundi trial
	BPM	CTL	BPM	CTL	BPM
DM¹	936.2	939.6	927	948.0	939.7
CL	32	111.7	93.9	115.0	87.3
Fiber	2.6	18.4	11.8	23.0	18.9
CP	461.4	520.4	504.9	526.4	508.7
Ash	132.5	89.8	102.7	90.2	103.2
NFE²	371.5	259.6	286.8	245.5	281.9
GE³	18.7	21.5	20.8	21.6	20.7
Yttrium oxide		7.1	4.5	11.0	5.9

Essential amino acids
Lysine	13.6	43	36.9	32.0	26.7
Arginine	24.8	36.9	33.4	36.4	30.9
Histidine	6.6	13.8	10.9	13.4	10.7
Isoleucine	18.1	23.1	20.9	24.8	22.5
Leucine	30.0	41.7	37.9	43.0	38.2
Valine	26.1	25.6	25.0	27.4	26.8
Methionine	10.4	11.2	11.0	12.0	11.4
Phenylalanine	17.2	25.4	22.9	26.2	23.1
Threonine	19.3	23.4	22.0	23.3	21.5

Non-essential amino acids
Alanine	32.9	29.4	30.7	29.2	30.3
Aspartic acid	37.0	59.2	52.2	58.3	50.3
Cystine	2.5	7.3	4.7	6.6	5.4
Glutamic acid	45.5	86.8	73.6	90.9	74.6
Glycine	19.7	31.1	30.0	26.1	24.6
Proline	16.3	33.6	32.6	25.4	22.0
Serine	13.9	26.3	22.3	26.2	21.3
Tyrosine	14.4	18.4	17.4	19.5	15.0

Fatty acids⁴
SFA	2.7	30.9	21.7	30.5	19.5
MFA	8.2	21.7	18.7	21.2	16.4
PUFA	0.0	38.8	25.8	38.2	24.5
LC-PUFA	0.0	20.3	12.9	19.6	12.8
Omega-3	0.0	21.9	14.1	21.3	13.8
Omega-6	0.0	16.8	11.7	16.9	10.7
Omega-7	2.0	5.4	4.5	5.4	4.1
Omega-9	0.0	13.8	9.9	13.2	8.8
TFA	10.9	104.6	76.2	102.9	68.9

¹ Dry matter in g kg^− 1.

² NFE: nitrogen-free extracts

³ Gross energy in MJ kg^− 1 DM

⁴ SFA: saturated fatty acids, MFA: mono-unsaturated fatty acids, PUFA: poly-unsaturated fatty acids, LC-PUFA: long-chain poly-unsaturated fatty acids, TFA: total fatty acids. Refer to Table A1 for the composition of fatty acid groups.

Enzyme assays

Three fish from each tank were sampled, and their entire digestive tracts were mixed with 10 ml of deionized water. Intestinal enzymes were extracted by homogenizing the mixture in a 15 ml Dounce homogenizer and centrifuging it at 21,000 × g for 30 minutes at 4°C. The supernatant was stored at − 80°C for further enzyme activity analyses. The protein concentration (in mg ml^− 1) of each sample was determined via the Bradford assay using standards of bovine serum albumin (Quick Start Bradford Protein Assay Kit, Bio-Rad 5000201). Protease activities were measured using standard colorimetric substrates and the methods described by Natalia et al. (2004), adapted for 96-well format using a microplate reader. Assays were performed at room temperature by mixing the crude extracts with substrates in 50 mM Tris, pH 7.4, in a total volume of 350 µl. The substrates used with trypsin, chymotrypsin, and leucine aminopeptidase were, respectively, 0.1 mM Nα-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA) + 20 mM CaCl₂, 0.1 mM N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide (SAPNA) + 20 mM CaCl₂, and 1.0 mM L-leucine-4-nitroanilide (LeuPNA) + 20 mM MgCl₂. Proteolytic reactions were followed over time by recording the absorbance at 410 nm. Lipase activity was measured using a commercial kit (Sigma-Aldrich, MAK046-1KT), where the production of glycerol from triglyceride by lipase was monitored using absorbance at 570 nm. One enzyme unit (U) was defined as one nmol of product produced per minute. For trypsin, chymotrypsin, and leucine aminopeptidase, the conversion of absorbance to nmol of p-nitroaniline was calculated using the molar extinction coefficient of 8,800 M^− 1cm^{− 1 14}. For lipase, the molar amount of glycerol was derived from a standard curve obtained by measuring the absorbance at 570 nm of known concentrations of glycerol. Specific activity was expressed as U mg^− 1 total protein present in the crude extract. The enzyme kinetic parameters, namely the maximum velocity (Vmax, in U mg^− 1) and the Michaelis–Menten constant (Km), were determined for trypsin, chymotrypsin, and leucine aminopeptidase. This was accomplished using concentration gradients of BAPNA, SAPNA, and LeuPNA as substrates.

Data collection

The mean values for the initial and final fish body weights (FBW, in g) were employed to compute both the body weight gain (BWG, in g) and the TGC for each tank. TGC served as a standardized measure of growth ¹⁵ and was assumed to be unaffected by variations in body weight, time intervals, and water temperature ^16,17. In this analysis, the TGC was calculated with a base value of 20 to enhance its stability, which is especially important in high-temperature conditions ¹⁸:

\(\:{TGC}_{a}=1000\text{*}\frac{{FBW}_{a}^{(1/3)}-\:{IBW}_{a}^{(1/3)}}{{\sum\:}_{\text{i}=0}^{51}({\text{T}}_{\text{i}}-20)}\) (Eq. 1)

where FBW_a and IBW_a are the final and initial average FBW (g) in tank a, and T_i (°C) is the water temperature on day i. The feed conversion ratio (FCR) in tank a was calculated as follows:

\(\:{FCR}_{a}=\left(\sum\:_{i=1}^{51}{TI}_{ai}/{qty\:of\:fish}_{ai})/{(FBW}_{a}-{IBW}_{a}\right)\) (Eq. 2)

where TI_ai is the tank feed intake (g day^− 1) in tank a on day i. The apparent digestibility coefficients (ADCs) (%) of DM, CP, CL, GE, nutrients, or nutrient groups in the experimental diets were calculated as follows:

\(\:{ADC}_{parameter}=\left[1-\left(\frac{{Y}_{Diet}\times\:\:{Parameter}_{feces}}{{Y}_{feces}\times\:\:{Parameter}_{diet}}\right)\right]\times\:100\) (Eq. 3)

where \(\:{Y}_{diet}\) and \(\:{Y}_{feces}\) are the yttrium content of the diet and feces, respectively, and \(\:{Parameter}_{diet}\) and \(\:{Parameter}_{feces}\) are the content of the nutritional parameter of interest (DM, CP, CL, GE, nutrient, or nutrient group) in the diet and feces, respectively. The ADCs of a given nutrient from BPM ingredient (ADC_ingr) were calculated as follows:

\(\:{ADC}_{ingr}=\left({ADC}_{test}{\times\:Nutr}_{test}-\left({ADC}_{ref}{\times\:Nutr}_{ref}\times\:Rb\right)\right)/\left(\left(1-Rb\right){\times\:Nutr}_{ingr})\right)\) (Eq. 4)

where ADC_test is the ADC of the diet containing the ingredient, ADC_ref is the ADC of the reference diet. Nutr_test, Nutr_ref, Nutr_ingr are the level of the nutrient of interest in the test diet, reference diet and the ingredient respectively ¹⁹. The ratio 1 − Rb at which the test ingredient was included in the test diet was determined based on the effective dilution of yttrium between the basal and test diets, according ²⁰.

Statistical analysis

To analyze fish performance and ADCs, one-way analysis of variance (ANOVA) was employed, and pairwise comparisons between species or diets were conducted using Tukey's honestly significant difference (HSD) multiple range test. An arcsine-square-root transformation was applied to the survival rate and ADCs. All kinetic parameters were obtained by subjecting the data to the non-linear regression methods described by the Michaelis–Menten equation: V = Vmax([S]/ (Km + [S])) (Eq. 5), where V is the velocity of the reaction, [S] is the substrate concentration, Vmax is the maximum velocity, and Km is the Michaelis–Menten constant. This methodology was preferred over the Lineweaver-Burk method to improve the accuracy of kinetic parameter estimates ²¹. For All statistical analyses were performed using R software ²², and effects were considered statistically significant at a significance level of P < 0.05.

Table 3. Fish performance of tilapia and barramundi fed experimental diets

	CTL	BPM
Tilapia trial
IBW	51.4 ± 4.1^a	45.6 ± 3.8^a
FBW	72.3 ± 4.6^a	59.6 ± 5.5^b
BWG	20.8 ± 1.3^a	14.1 ± 2.0^b
TFI	22.4 ± 0.0^a	22.4 ± 0.0^a
TGC	1.9 ± 0.1^a	1.4 ± 0.1^b
FCR	1.1 ± 0.1^a	1.6 ± 0.2^b
Surv	100.0	100.0

Barramundi trial
IBW	49.7 ± 0.6^a	49.2 ± 0.6^a
FBW	63.4 ± 1.4^a	60.6 ± 1.0^b
BWG	14.5 ± 0.8^a	12.1 ± 0.9^b
TFI	15.6 ± 0.0^a	15.6 ± 0.0^a
TGC	1.7 ± 0.1^a	1.4 ± 0.1^b
FCR	1.1 ± 0.1^a	1.3 ± 0.1^b
Surv	100.0	98.7 ± 2.3

Values are shown as means (n = 3, ±SD), and values with differing superscripts are significantly different (P < 0.05) for 28-day tilapia trial and 23-day for barramundi trial.

IBW: initial body weight (g); FBW: final body weight (g); BWG: body weight gain (g fish^-1); TFI: total feed intake (g fish^-1);TGC: thermal-unit growth coefficient in base 20; FCR: feed conversion ratio (g feed (as fed) / g wet weight gain); Surv: Survival rate (%)

Diet digestibility

Both species exhibited high thermal-unit growth coefficients (1.7-1.9) and low feed-conversion ratios (1.1) on control diets (Table 3). The diet ADC for protein was slightly but significantly lower in tilapia than in barramundi (P < 0.05) (Table 4), with values of 94.8 ± 0.6% and 95.8 ± 0.5%, respectively. This trend was consistent for all essential amino acids except lysine. Conversely, tilapia exhibited significantly higher digestibility for PUFA and omega-6 (P < 0.05), while other fatty acids had similar ADCs in both species. The incorporation of 30% BPM negatively impacted the diet digestibility of most nutrients it contains, such as amino acids and MFA, in both species (P < 0.05). The species*diet interaction was insignificant for almost all nutrients.

Table 4. Mean (n=3) apparent digestibility coefficients (%) of experimental diets

	Tilapia		Barramundi		Effects
	CTL	BPM	CTL	BPM	Species	Diet	Species*Diet
DM	83.6 ± 1.1^a	73.2 ± 2.0^b	81.4 ± 2.1^a	74.9 ± 1.9^b	Ns	***	ns
CP	94.8 ± 0.6^a	86.6 ± 1.9^b	95.8 ± 0.5^a	90.6 ± 0.4^c	**	***	ns
GE	87.5 ± 1.1^a	78.9 ± 1.5^b	84.9 ± 1.2^a	79.4 ± 1.9^b	Ns	ns	ns
CL	92.9 ± 1.3^a	86.3 ± 1.5^a	86.8 ± 3.6^a	84.2 ± 8.1^a	Ns	***	ns

Essential amino acids
Arginine	96.7 ± 0.5^a	91.9 ± 0.7^b	98.0 ± 0.2^c	94.2 ± 0.7^d	***	***	ns
Histidine	96.4 ± 0.6^a	92.0 ± 0.9^b	97.3 ± 0.4^a	93.3 ± 0.7^b	*	***	ns
Isoleucine	94.6 ± 0.9^a	91.4 ± 1.6^b	97.3 ± 0.6^c	94.3 ± 0.4^a	***	***	ns
Leucine	94.9 ± 0.8^a	92.2 ± 1.2^b	97.4 ± 0.6^c	94.9 ± 0.4^a	***	***	ns
Lysine	97.0 ± 0.5^a	94.6 ± 0.4^b	97.5 ± 0.3^a	94.5 ± 0.8^b	ns	***	ns
Methionine	94.8 ± 0.9^ab	91.4 ± 1.3^c	96.3 ± 0.6^a	93.3 ± 0.3^bc	**	***	ns
Phenylalanine	95.0 ± 0.8^a	91.9 ± 1.1^b	97.1 ± 0.7^c	94.2 ± 0.5^a	***	***	ns
Threonine	93.6 ± 0.8^a	88.3 ± 1.3^b	96.8 ± 0.8^c	92.9 ± 0.3^a	***	***	ns
Valine	94.5 ± 0.9^a	90.0 ± 1.5^b	95.6 ± 0.7^c	92.6 ± 0.5^ab	**	***	ns

Alanine	93.6 ± 0.9^a	87.5 ± 1.5^b	95.6 ± 0.5^c	91.6 ± 0.8^a	***	***	ns
Aspartic Acid	96.4 ± 0.5^a	92.3 ± 0.7^b	96.6 ± 0.3^c	93.7 ± 0.6^b	**	***	ns
Cystine	94.3 ± 0.8^a	85.1 ± 3.6^b	94.6 ± 0.5^a	85.0 ± 3.0^b	ns	***	ns
Glutamic Acid	97.3 ± 0.4^a	92.9 ± 0.8^b	98.3 ± 0.2^c	94.9 ± 0.4^d	***	***	ns
Glycine	95.2 ± 0.6^a	89.6 ± 0.6^b	93.8 ± 0.9^a	89.5 ± 1.1^b	ns	***	ns
Proline	91.3 ± 2.3^ab	87.8 ± 5.2^b	96.1 ± 0.5^a	93.6 ± 0.3^ab	**	ns	ns
Serine	95.0 ± 0.7^a	89.3 ± 1.4^b	97.0 ± 0.5^c	92.8 ± 0.8^d	***	***	ns
Tyrosine	95.1 ± 0.8^a	91.3 ± 1.0^b	97.2 ± 0.4^c	91.3 ± 0.6^b	**	***	*

TFA	97.3 ± 2.3^a	93.4 ± 1.8^a	92.7 ± 1.5^a	92.5 ± 2.2^a	*	ns	ns
SFA	97.1 ± 2.6^a	93.9 ± 2.4^a	92.3 ± 2.1^a	93.20 ± 2.7^a	ns	ns	ns
MFA	97.2 ± 2.6^a	93.12 ± 2.3^a	94.7 ± 0.8^a	92.5 ± 1.6^a	ns	*	ns
PUFA	97.6 ± 1.9^a	93.7 ± 0.8^ab	92.0 ± 1.6^b	92.1 ± 2.5^b	**	ns	ns
LC-PUFA	97.8 ± 2.6^a	96.2 ± 1.5^a	97.6 ± 0.1^b	98.0 ± 1.8^ab	ns	ns	ns
Omega 3	97.6 ± 3.1^a	96.7 ± 1.4^a	97.0 ± 0.8^b	98.1 ± 1.6^ab	ns	ns	ns
Omega 6	97.2 ± 1.3^a	90.3 ± 0.2^b	85.7 ± 2.7^b	84.5 ± 3.6^b	**	*	*
Omega 7	98.2 ± 3.1^a	96.0 ± 3.5^a	100.0 ± 0.0^a	100.0 ± 0.0^a	ns	ns	ns
Omega 9	96.9 ± 2.4^a	92.4 ± 2.0^a	92.7 ± 1.1^a	92.6 ± 1.6^a	ns	ns	ns

Values are shown as means ±SD (n=3), and values with differing superscripts are significantly different (P < 0.05); tested effects: * P < 0.05; ** P < 0.01; *** P < 0.001; ns: non-significant; when interaction was significant, the effects were separately tested; TFA: total fatty acids, SFA: saturated fatty acids, MFA: mono-unsaturated fatty acids, PUFA: poly-unsaturated fatty acids, LC-PUFA: long-chain poly-unsaturated fatty acids. Refer to Table A1 for the composition of fatty acid groups.

BPM digestibility

The ADCs of BPM for dry matter and protein (Table 5) were higher in barramundi (66.3 ± 5.8% and 87.5 ± 0.9%, respectively) than in tilapia (54.1 ± 5.1% and 71.8 ± 5.3%, respectively). This difference was primarily driven by higher ADCs for histidine, isoleucine, leucine, phenylalanine, threonine, and valine in barramundi.

Table 5. Apparent digestibility coefficients of the tested ingredients¹

	Tilapia	Barramundi
Dry matter	54.1 ± 5.1^a	66.3 ± 5.8^b
Crude protein	71.8 ± 5.3^a	87.5 ± 0.9^b

Essential amino acids
Arginine	87.4 ± 2.6^a	82.3 ± 2.0^a
Histidine	65.7 ± 3.9^a	94.7 ± 2.5^b
Isoleucine	78.0 ± 4.8^a	96.1 ± 1.1^b
Leucine	87.1 ± 3.8^a	96.5 ± 1.2^b
Lysine	100.0	100.0
Methionine	85.0 ± 3.6^a	89.6 ± 0.7^a
Phenylalanine	88.4 ± 3.9^a	99.1 ± 1.4^b
Threonine	76.3 ± 3.7^a	85.9 ± 0.8^b
Valine	72.9 ± 3.7^a	86.8 ± 1.2^b

Non-essential amino acids
Alanine	76.2 ± 3.6^a	83.2 ± 1.6^b
Aspartic Acid	87.0 ± 2.5^a	93.6 ± 1.8^b
Cystine	n.a.	100.0
Glutamic Acid	87.5 ± 3.4^a	100.0^b
Glycine	100.0^a	95.8 ± 3.1^a
Proline	100.0^a	96.2 ± 0.9^b
Serine	79.9 ± 5.7^a	90.8 ± 2.7^b
Tyrosine	87.9 ± 3.5^a	48.8 ± 1.5^b

Values are percentages shown as means ±SD (n = 3), and values with differing superscripts are significantly different (P < 0.05); In cases where apparent digestibility coefficients exceeded 100%, a practical assumption of absolute digestibility at 100% was applied.

Digestive enzyme activities and kinetics

Tilapia showed higher enzyme activities compared to barramundi for trypsin, chymotrypsin, and leucine aminopeptidase (Table 6) (P < 0.05). There was a significant interaction effect for chymotrypsin activity, indicating that the response to the BPM diet differs between species. The Km values indicate differences in enzyme affinities between species and diets. Tilapia exhibited higher enzyme affinity (lower Km) for trypsin in the BPM diet (4400.8 U vs. 3176.3 U), whereas barramundi showed higher affinity for leucine aminopeptidase in the BPM diet (Km = 59.8 vs. 70.0) (P<0.05). There was no significant effect of BPM incorporation on enzymes activities and affinities but significant interaction effects suggest that the relationship between diet and enzyme kinetics varies between species (Fig. 1).

Table 6. Mean specific activity¹ and Michaelis–Menten constant of trypsin, chymotrypsin, and leucine aminopeptidase enzymes in the gut of fish fed experimental diets²

	Tilapia		Barramundi		SEM	Species	Diet	Species*Diet
	CTL	BPM	CTL	BPM
Activity
Tryp	320.0^a	296.8^a	13.7^b	19.0^b	55.8	***	ns	ns
Chym	4400.8^a	3176.3^b	228.4^c	300.1^c	633.5	***	ns	*
Lap	30.1^a	27.9^a	13.1^b	15.5^b	5.2	***	ns	ns

Km
Tryp	11.8^a	4.8^b	15.1^a	22.6^c	2.5	**	ns	***
Chym	240.0^a	205.8^a	425.7^b	530.4^b	52.4	***	ns	*
Lap	132.2^a	133.0^a	70.0^b	59.8^c	3.94	***	ns	***

¹ One enzyme unit (U) was defined as one nmol of product produced per minute.

²Values (U mg^-1 protein) are shown as means ±SD (n = 3), and values with differing superscripts are significantly different (P < 0.05); Tested effects: * P < 0.05; ** P < 0.01; *** P < 0.001; ns: non-significant; when interaction was significant, the effects were separately tested; Km: Michaelis–Menten constant in µM; Tryp: trypsin, Chym: chymotrypsin, Lap: leucine aminopeptidase.

The lines through the data points represent the least-squares regression fit of the Michaelis–Menten equation (Eq. 7). Enzyme reaction rates are shown in U mg protein^-1. Plotted points represent mean ± SD values (n = 3). Substrates used with trypsin, chymotrypsin and leucine aminopeptidase were BAPNA, SAPNA and LeuPNA respectively.

Water quality

The daily water quality during the tilapia trial was as follows: temperature averaged 28.1 ± 0.5°C, dissolved oxygen averaged 7.0 ± 0.5 mg L^-1, salinity averaged 5.3 ± 0.5 ppt, pH averaged 6.3 ± 0.5, and total ammonia remained below 0.25 mg/L. The daily water quality during the barramundi trial was as follows: temperature averaged 28.7 ± 0.5°C, dissolved oxygen averaged 7.1 ± 0.1 mg L^-1, salinity averaged 5.0 ± 0.1 ppt, pH averaged 6.9 ± 0.2, and total ammonia remained below 0.0 mg/L.

Diet apparent digestibility in two species

In this study, the ADCs for both barramundi and tilapia were determined across two consecutive trials conducted at a water temperature of 28°C and 5 ppt salinity. The ADC for protein was slightly but significantly lower in tilapia than in barramundi (P < 0.05) (Table 4) and for all essential amino acids except lysine. In this study, the protein content of the control diet was 43.2 %. While this level falls within the requirement range for both species, it may have been too high for tilapia or too low for barramundi, potentially explaining the slight difference observed. However, more research is needed to define the nutrient requirements of this O. mossambicus tilapia, which are not well-defined ⁷. A longer acclimatization period could also improve the accuracy of diet ADC estimation, as evidenced by more stable results after a 17-day acclimatization period when testing BPM in Atlantic salmon (Salmo salar) ²³. Conversely, tilapia exhibited significantly higher digestibility for PUFA and omega-6 (P < 0.05), while other fatty acids had similar ADCs in both species. Notably, Salini et al. (2016) reported that total lipid digestibility in barramundi juveniles was negatively affected by omega-6 fatty acid enrichment from soy lecithin. In the current study, soybean lecithin was used as a fatty acid source, which could potentially explain the observed limited digestibility associated with this ingredient. However, investigating the specific effects of soy lecithin fell beyond the scope of our study objectives.

Ingredient apparent digestibility

Estimating nutrient digestibility is a crucial first step in aquaculture feed formulation ²⁵ and this study represents the earliest attempts to characterize BPM digestibility in non-salmonids species. The measured ADC of BPM (Table 5) for protein in barramundi (87.5%) was higher than those estimated for BPMs produced from natural gas in Atlantic salmon (81.9%, 78.4%) ^26,27. However, the measured ADC of BPM for protein in O. mossambicus tilapia was lower (71.8%) than those previously cited. The lower digestibility of BPM protein tilapia, driven by lower ADC for specific amino acids, cannot be attributed to a negative impact on digestive enzyme activity or affinity (Table 6). These results highlight the potential variations in BPM digestibility across different fish species.

Enzyme activity and kinetics

There was no significant effect of BPM incorporation on enzymes activities and affinities which is a good indication that the BPM did not contain trypsin, chymotrypsin, or leucine aminopeptidase inhibitors (Fig. 1). However, significant interaction effects suggest that the relationship between diet and enzyme kinetics varies between species (Table 6, Fig. 1). While explorations of the effects of dietary composition on fish digestive enzymatic activity are still limited, Santigosa et al. (2008) observed a reduction in protease activity in rainbow trout (Onchorynchus mykiss) when plant protein sources were introduced into the diet. Recent studies exploring the impact of dietary administration of functional feed additive on digestive enzyme activity revealed contradictory effects on various fish species ²⁹. In the current study, a strong species effect was evident, with tilapia exhibiting higher enzyme activities compared to barramundi, (Table 6) when the affinity differences between species were more complex.

High trypsin affinity

The measured Km values indicate high affinity of digestive trypsin for its substrate in both barramundi (Km = 15.1 nM) and tilapia (Km = 11.8 nM), significantly higher than those reported for other fish species (Km = 51.0 – 387.0 nM). Specifically, trypsin affinity in our study is 3-30 times higher than in other species, such as striped seabream (Lithognathus mormyrus) (Km = 290 nM) ³⁰, grey triggerfish (Balistes capriscus) (Km = 68.0 nM) ³¹, white croaker (Micropogonias furnieri) (Km = 81.0 nM) ³², Monterey sardine (Sardinops sagax caerulea) (Km = 51-130 nM) ^33–35, smooth Hound (Mustelus mustelus) (Km = 387.0 nM) ³⁶, and skipjack tuna (Katsuwonus pelamis) (Km = 220.0 nM) ³⁷. This variability could stem from differences in measurement protocols or represent an adaptive response of species to diverse feeding regimes, or domestication, warranting further research. Over the past years, trypsin from fish viscera also shown biotechnological potential as a source of proteolytic enzymes in food processing ³⁰. The high affinity of trypsin from barramundi and saline tilapia suggests that processing byproducts from these species could be of interest for producing industrial enzymes.

Author Contribution

R. L. was responsible for research conceptualization, methodology, formal analysis, investigation, data curation, writing, supervision, and funding acquisition. C. W. contributed to research conceptualization, methodology, investigation, data curation, and formal analysis. T. A. B. S. and M. K. provided resources and conducted investigations. W. C., L. T., and J. N. were involved in the investigation. C. S. L. handled methodology, investigation, project administration, and data curation.

Acknowledgement

The authors express gratitude to Tuty Adilah Binte Sapri and Meng Koon for their commitment to the breeding program of Oreochromis mossambicus from 2004 to 2022 in Singapore, and to Temasek Life Sciences for providing long-term funding for the breeding program.

Data Availability

Data is available upon request from the corresponding author.

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Bacterial protein meal digestibility, digestive enzyme activity and kinetics in barramundi (Lates calcarifer) and tilapia (Oreochromis mossambicus) juveniles

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