Evaluation of corn steep liquor on the digestibility and growth performance of juvenile common carp strain Sinyonya Evaluasi corn steep liquor terhadap kinerja kecernaan dan pertumbuhan juvenil ikan mas Sinyonya

This study aimed to observe the digestibility performance, digestive enzyme activity, and growth performance of the common carp using corn-steep liquor (CSL). The reference diet used commercial feed with 30% protein, and the test diet used CSL at 0% (CSL0), 10% (CSL10), 20% (CSL20), and 30% (CSL30). Common carp (9.38 ± 0.73 g) were stocked at 10 fish per aquarium. Feeding was performed until apparent satiation thrice daily for the 45-day maintenance period. The study used a completely randomized design with four treatments and four replications. The results showed that CSL contained 44.04% protein and an essential amino acid index of 0.80. The additional dose of CSL reduced feed pH and stomach pH compared to the control due to the lactic acid content. In addition, CSL elevated the activity of protease, amylase, and lipase enzymes in the digestive tract of common carp. By applying 20% dose in formulated diet, CSL could increase the total digestibility, ingredient digestibility, protein digestibility, lipid digestibility, and energy digestibility. The high feed digestibility value in the CSL20 treatment was due to the fish ability to utilize carbohydrates and fats as non-protein energy sources, thus producing higher final weight, daily growth rate, and feed conversion ratio. The results of this study indicate that CSL can be used as a source of plant protein for the common carp diet, whereas an administration of CSL at 20% in diet improves the digestibility performance, digestive enzyme activity, and growth performance of common carp.


INTRODUCTION
Common carp (Cyprinus carpio) is one of the economically important cultured fish commodities in Indonesia.In 2015 to 2020, the common carp production significantly contributed to Indonesia's fisheries production, with a growth rate of 9.60%.In 2020, the production reached 560,670 tons (KKP, 2022), which included the common carp strain Sinyonya.The common carp strain Sinyonya is an excellent fish commodity in Banten Province due to high economical value.However, producing the common carp strain Sinyonya has drawbacks in fish seed availability and cheap protein ingredients for aquafeed requirements to support their growth.
The development of fish culture business increases the fish demand and feed ingredient from protein source.The combined data from livestock feed entrepreneurs (GMPT) shows an increased feed production in 2008-2016 by 65%, with a total production of 1.63 million tons in 2016.The feed dependency poses a high risk to aquaculture business sustainability, whereas approximately 91.2% of the production costs for common carp culture are absorbed by feed cost (Suprayudi, 2018).To ensure sustainable aquafeed production, alternative feed ingredients are essential as protein source that composes appropriate nutrient contents for fish requirement with adequate quantities and availability.
Potential aquafeed ingredient that can be developed further comes from waste or agroindustrial by-product to regulate efficient resource utilization and support sustainable aquaculture practices (Caruso, 2015).NRC (1993) recommended the protein requirement for common carp based on age size, namely 43-47% for seeds, 37-42% for fingerlings and subadults, and 28-32% for adults and broodstocks.To fulfill the protein requirement, several studies have shown that plant-based materials can be used as a protein source in formulated diet for fish (Magbanua & Ragaza, 2024).The agroindustrial by-products that have been applied as plant-based protein aquafeed ingredient are palm kernel meal (Wattanakul et al., 2021), dried distiller grains with solubles (Chatvijitkul et al., 2016), corn gluten meal (Bu et al., 2018), corn steep liquor (Sukhanandi & Bhatt, 2016), and corn steep powder (CSP) (Hermawan et al., 2021).
According to Wattanakul et al. (2021), replacing dehulled soybean meal with fermented palm kernel meal by 50% could improve the growth performance, feed utilization, and digestive enzyme activities.Chatvijitkul et al. (2016) reported that the application of dried distiller's grains with solubles at 300 g/kg feed could regulate the growth performance of hybrid Nile tilapia.Hermawan et al. (2021) also stated that the application of CSP at 20% inclusion in formulated diet could improve the digestive enzyme, digestibility, and growth performance of fish.Another potential aquafeed ingredient that has never optimized as plant-based protein ingredient is corn steep liquor (CSL).CSL or corn steep water is a wet milling by-product of corn starch production formed as a brown viscous liquid that contains protein, minerals, vitamins, reducing sugars, organic acids, and enzymes (Tan et al., 2016).
CSL is highly available as the production could reach 1000 tons/day (Tereos, 2016).Due to relatively high protein and energy contents with low fiber content, CSL is often utilized as potential feed for ruminants (Jiriaei et al., 2020), while minerals and vitamin B in CSL can also be used as a feed binder (Azizi-Shotorkhoft et al., 2016).Chovatiya et al. (2011) showed that CSL could replace fish meal by 75% in formulated diet for Labeo rohita without inhibiting the growth, thus improving the meat nutrient contents, such as protein and lipid.The utilization of plant-based protein material as an aquafeed ingredient often has problems dealing with the anti-nutritional properties and imbalance amino acid profiles.
These anti-nutritional properties tend to disrupt the digestive function, nutrient metabolism, and fish production (Omnes et al., 2017).However, CSL is free from phytic acid, aflatoxin, and tripsininhibitor.Low anti-nutritional properties in CSL is due to lactic acid fermentation by Lactobacillus sp.during the soaking process in corn wet-milling to produce corn starch, thus reducing the phytic acid content (Rahimi et al., 2017).Nevertheless, there have been no studies related to the application of CSL as an aquafeed ingredient in common carp strain Sinyonya.Therefore, this study investigated CSL as plant-based protein ingredient to improve digestibility, digestive enzyme activities, and growth performance.

Study design
This study used a completely randomized design with four treatments and four replications in each treatment.The CSL was obtained from PT Tereos FKS Indonesia, Cilegon, Banten.The composition of CSL was also determined, including nutrient contents (proximate analysis), amino acids, acid detergent fiber (ADF), neutral detergent fiber (NDF), aflatoxin, tannins, saponins, trypsin-inhibitor, and lactic acid.

Diet formulation
The diet was formulated using the commercial feed as a reference diet with 32% protein content.Test diets were formulated with different CSL concentrations at 0, 10, 20, and 30% (Carvalho et al., 2016).All diets were produced by grinding the commercial feed, adding different concentrations of test ingredients and Cr2O3 at 0.5 % as a digestibility test marker (Mmanda et al., 2020).All ingredients were mixed and formed as pellet (repelleting method).All diets were analyzed their nutrient contents through proximate analysis.The diet formulation for digestibility test is presented in Table 1.

Fish rearing and data collection
Common carp strain Sinyonya as objects in this study were weighed at averagely of 9.38 ± 0.73 g.Before being reared for 45 days, acclimatization process was performed for a week.Fish were reared in 16 units of aquarium at 60×50×40 cm 3 volume equipped with aeration system for 24 hours.Fish were stocked at 10 fish per aquarium.Before feeding, fish were fasted for 24 hours.Fish weight was measured on the initial and final rearing period.
The diets were fed three times a day at 08.00, 12.00, and 16.00 WIB (GMT+7) until apparent satiation.Fish feces were collected after four days of feeding (Watanabe, 1988).Feces were collected in a bottle by siphoning an hour after feeding.Collected feces was kept in a freezer at -20°C.

Proximate analysis
Proximate analysis performed in this study was composed of protein, ash, crude fiber, and moisture contents of CSL, diets, and feces.Protein content was determined using the Kjeldahl method.Soxhlet method and furnace-heating at 600°C were applied to determine lipid and ash contents.Meanwhile, samples dissolved in strong acid-base were performed to measure crude fiber and moisture content was determined through oven-heating at 110°C for 4 hours (AOAC, 2012).As comparative data, the corn steep powder (CSP) proximate analysis results by Hermawan et al. (2021) are also presented.

Amino acid analysis
Amino acid analysis of CSL used the HPLC (high performance liquid chromatography) method that contained four steps, namely sample hydrolysate production, drying, derivatization, and amino acid injection (AOAC, 2012).As comparative data, the amino acid analysis results of soybean meal (SBM) by IAAFD (2023) are also presented.

Digestive enzyme analysis
The digestive enzyme activity was measured on the 45 th day of rearing period.During the final day of rearing period, fish were fasted for 24 hours, and three fish were taken from each aquarium.Fish were sedated with Tricaine methane sulfonate (MS-222, Syndel, USA) at 0.15 g/L (Al-Taee et al., 2021).The digestive enzyme activity from the fish digestive tract was determined by separating the digestive organ from the fish body.The intestine was collected and homogenized in 0.15 M NaCl (40 mg tissue/mL).The sample was centrifuged at 10,000 ×g for 10 minutes at 4°C.The sample was dissolved in distilled water 10 times to obtain crude enzyme extract kept in -20°C for further enzyme activity analysis.The protease enzyme was measured using casein as a substrate and tyrosine as a standard (Bergmeyer et al., 1983).Lipase enzyme was measured using plant-based oil as a substrate (Linfield et al., 1984).Amylase enzyme was determined using starch as a substrate and maltose as a standard (Bernfield, 1955).

Digestibility analysis
Feces were collected after four days of feeding the diets (Watanabe, 1988).The Cr2O3 contents in diets and feces were performed by drying and absorbance reading in a spectrophotometer at 350 nm wavelength (Takeuchi, 1988).

pH diets and digestive tract
The pH level was measured by mixing 5 g of diet sample in 50 mL of distilled water until homogenous with magnetic stirrer for a minute (Li et al., 2015).The pH level in stomach was measured after 2 hours of feeding and the pH level in the intestine was measured after 6 hours of feeding (Castillo et al., 2014).The stomach and intestine contents at 5 g each were dissolved in 50 mL of distilled water (Li et al., 2015).The pH levels of diets, stomach, and intestine were measured with pH meter.

Digestibility and growth performances
Digestibility performance was determined based on Takeuchi (1988), namely total digestibility, ingredient digestibility, protein digestibility, lipid digestibility, and energy digestibility.Total digestibility was measured using the formula: Feed conversion ratio (FCR) was calculated with the formula: The growth performance of the fish was determined on the final rearing period at 45 th day.Parameters for growth performance were composed of feed intake (FI), specific growth rate (SGR), and feed conversion ratio (FCR).Feed intake was measured with: Specific growth rate (SGR) was calculated using the formula:

Data analysis
The SR, SGR, FCR, total digestibility, ingredient digestibility, protein digestibility, lipid digestibility, energy digestibility, pH diet, pH digestive tract, and digestive enzyme activities were all analyzed using a one-way ANOVA at 95% confidence level and continued with the Duncan's test.All statistical analyses used SPSS 24.0 software.

CSL Composition
The proximate analysis and anti-nutritional properties of CSL are presented in Table 2.The protein content in CSL is 44.40%.The total energy in CSL is twice higher than CSP.Nevertheless, the CSL contains higher tannins and lower saponins than CSP.The pH level of CSL is acidic with the lactic acid content of 20.36%.
The amino acid composition of CSL is presented in Table 3.The EAAI of CSL is 0.8 and lower than soybean meal (SBM).The analysis results indicate that CSL has no cysteine amino acid and generally has lower amino acid contents than SBM, except tyrosine amino acid.

Growth performance
Growth performance of common carp strain Sinyonya is presented in Table 4.The results showed that the growth performance of CSL20 treatment was higher than other treatments  (P<0.05).The FI, FCR, and SGR data increased by CSL20, then decreased along with the elevated dosage of CSL.

Digestive performance
5Data on pH levels of diet, intestine, and stomach are presented in Table 5.The results showed that the pH levels of diet and stomach significantly decreased on the CSL20 and CSL30 treatments (P<0.05).However, the application of CSL had no significant difference on pH level of intestine (P>0.05).
The enzymatic activities of protease, lipase, and amylase are presented in Table 6.The results showed that protease, lipase, and amylase activities significantly increased by CSL20, then decreased along with the elevated dosage of CSL (P<0.05).
Digestibility levels, namely total, ingredient, protein, lipid, and energy digestibility are presented in Table 7.The results showed that the CSL20 treatment obtained the highest total, ingredient, lipid, and energy digestibility compared to other treatments (P<0.05).Digestibility increased due to the application of CSL, but then decreased after applying CSL20 treatment.

Discussion
The results present that the CSL protein content is 44.04%, which was similar to Azizi-Shotorkhoft et al. (2016) and Ullah et al. (2018) at 40-48%.The protein content in CSL is higher than other plant-based protein sources from agroindustrial by-products, such as corn steep powder (Hermawan et al., 2021), DDGS (distillers dried grains with solubles) at 26-28% (Abouel et al., 2021;Allam et al., 2020), rubber seeds at 21.6% (Yusuf et al., 2016) and palm oil meal at 17.93% (Sangavi et al., 2020).The essential amino acid index of CSL (0.8) is lower than CSP, but can still be categorized as a raw material with a good quality protein source as a source of plant-based protein ingredients for aquafeed (Bunda et al., 2015).
Table 5. pH levels of diet, stomach, intestine in common carp strain Sinyonya.
The presence of lactic acid in CSL20 and CSL30 significantly decreased the pH levels of diet and stomach.In the digestive tract, lactic acid is known to reduce gastric pH and inhibiting the active transport of excess internal protons, which requires cellular adenosine triphosphate (ATP) consumption, leading to cellular energy depletion (Vieco-Saiz et al., 2019).Lactic acid bacteria found in the fish digestive tract cause bacterial balance, thereby increasing feed digestibility by fermenting carbohydrates through an enzymatic reaction and decreasing the pH level (Wang et al., 2021).Hermawan et al. (2021) also showed that the application of CSP that contained lactic acid up to 30% in formulated diet could reduce the gastric pH from 4.67 to 4.25.
CSL has zero crude fiber content.Crude fiber content in feed below 15.5% can reduce the nutrient intake, increase digestion and absorption capacity, improve feed conversion ratio and digestibility coefficient, support growth performance and fish health (Sun et al., 2019;Bonvini et al., 2018;Kamarudin et al., 2018).In addition, low crude fiber will increase intestinal peristalsis, that can help digest and absorb nutrients, besides increasing the degradation rate of chemical compounds in feed (Manullang et al., 2018).The enzyme activity profiles in the fish digestive tract is an indicator of the fish capability to digest and utilize the nutrients in feed.
This study showed that CSL in formulated diet could increase the digestive enzyme activity of common carp strain Sinyonya, except protein digestibility, which was insignificantly different with CSL10 and CSL20 treatments.These results are similar to Hoseini et al. (2023), who showed that lactic acid administration to common carp could increase the intestinal amylase, lipase, trypsin, and chymotrypsin enzymes by altering the intestinal microbiota.Organic acids have no significant effect on digestive enzymes in the pancreas (Huan et al., 2019;Yao et al., 2019).However, the anti-nutritional properties in plantbased protein ingredient for aquafeed can decrease digestive enzyme activities in the intestine (Barlaya et al., 2021;Willora et al., 2022).
The presence of phytic acid inhibits the activity of protease, lipase, and amylase.However, CSL has no phytic acid content.In addition, beneficial bacteria can secrete digestive enzymes in the fish intestines, reducing antinutritional properties and suppressing their negative effects on digestive enzymes (Assan et al., 2022).Karatas et al. (2023) reported that the presence of lactic acid bacteria that produced phytase could break down phytate level in feed, thereby increasing the activity of digestive enzymes.
Energy digestibility in the CSL20 treatment had a higher value than other treatments, which indicates the fish ability to utilize carbohydrates and lipids as non-protein energy sources through regulating the activity of amylase and lipase.According to Liao et al. (2015), digestibility is influenced by the presence of enzymes and activity level of digestive enzymes in digestive tract.Halver (1989) stated that the energy digestibility of fish is 70% for grain-based ingredient materials, while animal-based ingredients is 85%.This study showed that the energy digestibility of feed using CSL is higher than the established standards.
Decreased crude fiber, increased protein level, and decreased antinutritional properties cause the increased digestibility level (Suprayudi et al., 2017).Meanwhile, the composition and quality of macronutrients in feed, feed production method, and environmental factors, such as water temperature, dissolved oxygen content, and stocking density are factors that influence energy digestibility in fish (Haidar et al., 2016).The amount of feed intake and energy digestibility in fish will be interrelated as fish consume feed to gain energy they need.Feed intake is affected by the feed palatability based on the aquafeed ingredient composition.
Low nutrient digestibility in feed indicates reduced energy availability that can be utilized by fish (Wulaningrum et al., 2019).Increasing the CSL concentration in formulated diet causes an increased FI in the CSL treatments.Feed conversion determines the function of feed in supporting fish growth, whereas the lower the value, the more effective the feed utilized (Fry et al., 2018).Moreover, feed conversion also indicates the way the feed is digested, as the amount of feed intake that can be absorbed by the body before being converted into body biomass (Afifah et al., 2021).
The CSL20 treatment had the lowest feed conversion, which means that the CSL20 formulated diet is easier to digest, allowing good nutrient absorption in digestive tract.In contrast, the CSL30 treatment had a higher feed conversion rate than other treatments.This condition occurred due to low digestibility levels in the CSL30 treatment, which reduced the nutrient availability for subsequent metabolic activities.A similar condition was also reported by Chovatiya et al. (2011), which mentioned that increasing the dose of CSL could reduce the FCR value in Labeo rohita fish.The lactic acid content in CSL was thought to influence fish growth performance, which was in accordance with Elala et al. (2015), who reported that increasing the organic acids in feed could improve the growth performance of fish by regulating the digestive enzyme activities and protein digestibility.
The results indicate that CSL can be used as a source of plant-based protein ingredient for aquafeed that improves growth performance in common carp strain Sinyonya.The application of CSL in formulated diet can improve the fish health status based on the blood profiles and phagocytic activity.Blood is responsible for transporting oxygen, carbon dioxide, nutrients, hormones, metabolisms, and antibody production (Aruldoss et al., 2014).Hemoglobin and HR values decreased on the CSL20 and CSL30 treatments, but these values could still be tolerated by the fish, due to optimal values of growth performance and feed conversion ratios at these doses.

CONCLUSION
CSL is a corn starch byproduct from agroindustrial processing with a protein content of 44.04%, pH 4.50, and 20.36% of lactic acid.This plant-based ingredient is free from antinutritional properties, such as aflatoxin, phytic acid, and trypsin inhibitors.The essential amino acid index value of CSL is 0.8, which indicates as a useful protein ingredient source.Therefore, CSL can be used as a plant-based protein ingredient in aquafeed for common carp strain Sinyonya, based on the increased digestive enzyme activity, digestibility performance, and growth performance.The application of CSL at 20% in formulated diet is recommended for further production of common carp strain Sinyonya feed.
Index (EAAI) was calculated based on Kirimi et al. (2020): Note: aa1 = Essential amino acid in the ingredient AA = Essential amino acid requirement n = Total evaluated amino acids Survival rate (SR) was determined at the final rearing period with the formula: = reference diet; CSL10 = CSL 10%; CSL20 = CSL 20%; CSL30 = CSL 30%.FI = feed intake; FCR = feed conversion ratio; SGR = specific growth rate; SR = survival rate.Different superscript letters behind the average value (± SD) on the same line present a significant difference (P<0.05).

Table 1 .
Diet formulation and proximate composition.

Table 2 .
Proximate analysis and anti-nutritional properties of CSL.

Table 3 .
Amino acid and essential amino acid index of CSL and SBM.

Table 4 .
Growth performance of common carp strain Sinyonya on different corn steep liquor concentrations.

Table 6 .
Enzyme activities of common carp strain Sinyonya on different corn steep liquor concentrations.

Table 7 .
Digestibility levels of common carp strain Sinyonya on different corn steep liquor concentrations.