Feeding diets composed of low level microparticle protein derived from fish and soybean meals and using organic calcium added with Lactobacillus acidophilus or citric acid on intestinal condition and performance of broilers

Yoseph Avian Saputra, Nyoman Suthama and Bambang Sukamto

Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang 50275, Central Java, Indonesia
nsuthama@gmail.com

Abstract

The present research was aimed to evaluate the combination of microparticle protein and organic calcium composed diet added with Lactobacillus acidophilus or citric acid on intestinal condition and performance of broiler. One hundred and sixty birds of 14 day-old broiler with body weight of 346 ± 9.5 g were randomly divided into 5 treatments and 4 replications. Treatments tested were CP21: diet with 21% intact protein; CP18+1.2LA: diet with 18% intact protein + 1.2 ml Lactobacillus acidophilus; CP18+1.2CA: diet with 18% intact protein + 1.2% citric acid; MCP18+1.2LA: diet with 18% microparticle protein using organic calcium + 1.2 Lactobacillus acidophilus; MCP18 + 1.2CA: diet with 18% microparticle protein using organic calcium + 1.2 citric acid. Parameters observed were length and relative weight of the intestine; jejunal villi length, lactic acid bacteria (LAB) and coliform populations; ileal protein digestibility; cecal short chain fatty acids (SCFA); meat protein mass (MPM), average daily feed intake (ADFI), final body weight (FBW), daily body weight gain (DBWG) and feed conversion ratio (FCR). Data were analyzed by analysis of variance and continued to orthogonal contrast test. Low protein diets composed of intact protein (CP18) and microparticle (MCP18) increased (p <0.05) relative weight of duodenum, jejunum and colon, cecal propionic acid production, colonic length and meat protein mass, but decreased ( p <0.05) ADFI, FBW and DBWG as compared to CP21. The MCP18 treatment increased (p <0.05) acetic, propionic and butyric acids production, and meat protein mass, but decreased (p <0.05) jejunal coliform population, ADFI, FBW, DBWG and FCR as compared to the CP18. In conclusion, diet composed of microparticle protein at low level and organic calcium added with additives ( Lactobacillus acidophilus or citric acid) are able to maintain the balance of intestinal microflora, increase intestinal development, SCFA production and meat quality indicated by meat protein mass, but decrease ADFI, FBW and DBWG with the same FCR.

Keywords: acidifier, particle size, probiotic, SCFA

Introduction

The current genetic development of modern broilers requires the supply of high amount and balanced protein to support rapid growth. Feeding high dietary protein level is common way to meet those needs. However, high protein content in the diet causes an increase in the amount of protein that possibly undigested in the intestine. This condition is possibly useless because undigested protein that enters the hind gut and is fermented by intestinal microflora (Qaisrani et al 2015). Protein fermentation products in the hind gut such as amines, indoles, phenols, cresol and ammonia have a negative impact on host health (Apajalahti and Vienola 2016). On the other hand, to overcome the problem as describe above, decreasing dietary protein level is possible, but lack of protein supply negatively affect performance and morphology of the intestine as it has been stated by Incharoen et al (2010); Laudadio et al (2012); Abbasi et al (2014). This becomes a major concern in the current development of broiler feed industry. Some previous studies demonstrated that when feeding low protein diet should be better to be combined with exogenous proteases (Cowieson et al 2016; Law et al 2018; Ndazigaruye et al 2019), prebiotics, probiotics and organic acids (Houshmand et al 2012 ; Mahmoud et al 2017; Ravangard et al 2017).

Feeding low level protein diet would be more effective when protein source ingredients are processed to become a microparticle protein using ultrasound methods. Ultrasound is a new technology in food processing (Yang et al 2015). Meanwhile, the use of ultrasound methods in the processing of animal feed ingredients is still rarely conducted. Microparticle protein as a result of ultrasound treatment containing broken peptide bonds, thereby, increasing the functional properties of protein (Huang et al 2017). Microparticle protein have better protein solubility (Hu et al 2013) and smaller protein sizes but without changing the molecular weight (O'Sullivan et al 2016). Suthama and Wibawa (2018) reported that protein source ingredients, such as fish meal and soybean meal, that processed become microparticle proteins using ultrasound transducer methods increased protein digestibility, amino acids and Ca retention in broilers. Protein and calcium are interrelated each other in the process of absorption through the compound called "calcium-binding protein (CaBP)". Therefore, the use of diet composed of microparticle proteins and organic calcium in broiler is interesting to be conducted. In case of the use of eggshells as organic calcium source, the previous study indicated that performance of laying hens was better than that used of limestone (Kismiati et al 2012).

In the present study, diet composed of microparticle protein at low level was combined with probiotic or acidifier in relation to intestinal condition and performance of broiler. It has been previously reported that probiotic (Alloui et al 2013; Sugiharto 2016; Cholis et al 2018; Yuanita et al 2019), and probiotic combined with dahlia inulin (Purbarani et al 2019) could increase beneficial bacteria in the intestine and suppress the development of pathogenic microorganism. While, acidifier decreased the pH of the digestive tract, thereby, increased lactic acid bacteria and lowered coliform in the small intestine (Yakhkeshi et al 2014). Beneficial microflora in the digestive tract can ferment either low molecular weight carbohydrates, such as oligosaccharides of soybean meal or inulin to produce "short chain fatty acids (SCFA)" which affect the development of the digestive tract its self. This phenomenon was indicated by an increase in ileal weight and villi-crypt ratio due to the use of probiotics (Olnood et al 2015) and an increase in the length of the villi and the crypt depth of the duodenum, jejunum and ileum caused by the use of acidifier (Nourmohammadi and Afzali 2013). The increased villi length, crypt depth or villi-crypt ratio can be used as an indication of improving intestinal absorptive area and support the absorption of nutrient, especially protein.

Therefore, the present study was conducted to evaluate the effect of feeding microparticles protein at low level and calcium organic combined with additives, namely, Lactobacillus acidophilus or citric acid on intestinal condition and performance of broiler.

Materials and methods

Experimental diet preparation

There were three types of experimental diets, namely, diet with 21% intact protein (CP21), diet with 18% intact protein (CP18) and diet with 18% microparticle protein and organic calcium (MCP18). The composition and nutrient content of the experimental diets are shown in Table 1. The diets were made in the form of pellet prior to feeding trial. Microparticle protein was prepared by grinding protein source feedstuffs (soybean meal and fish meal) into fine particles and continued to ultrasound transducer (ultrasonic bath) treatment according to Suthama and Wibawa (2018). The particle size obtained was 1.78 μm and 0.531 μm for fish meal and soybean meal, respectively, after it was measured using Nano Particle Analyzer (SZ-100, Horiba Ltd., Japan). Organic calcium was obtained from eggshell.

The experimental diets were added with additive of Lactobacillus acidophilus or citric acid based on the treatments that will be described in experimental animal and design section. Pure cultures of Lactobacillus acidophilus FNCC 0051 were obtained from Food and Nutrition Study Center, Gadjah Mada University and were cultured to have a population of 10⁸ cfu/ml. Citric acid in the form of powder was obtained from commercial products (PT. Budi Acid Jaya) at the nearest chemical store.

Experimental animal and design

A total of 200 birds of one-day old unsexed broiler of Cobb strain were used in the present study. The birds were given commercial diet until 14 days old prior to feeding experimental diet. When the broiler were 14-day old, 160 broiler with body weight of 345 ± 27.4 g were randomly divided into 20 treatment units and started feeding experimental diets. The birds were kept in colony cages until 21-day old and than they moved to individual batteries until 42-day old. Broiler received a small portion (±25 g) of experimental diets with additives according to treatment every morning before giving other portion to fulfill daily feed requirement.

This study was arranged in completely randomized design (CRD) with 5 treatments and 4 replications, with 8 bird each. Dietary treatments tested were CP21: diet with 21% intact protein; CP18+1.2LA: diet with 18% intact protein + 1.2 ml Lactobacillus acidophilus; CP18+1.2CA: diet with 18% intact protein + 1.2% citric acid; MCP18+1.2LA: diet with 18% microparticle protein using organic calcium + 1.2 Lactobacillus acidophilus; MCP18 + 1.2CA: diet with 18% microparticle protein using organic calcium + 1.2 citric acid.

Parameter and Statistical Analysis

Average daily feed intake (ADFI) and final body weight were recorded during the study. Daily body weight gain was obtained from final body weight reduced by initial body weight and then divided by the total of observation day. Feed conversion ratio was calculated from the total amount of consumed feed during the study divided by cumulative body weight gain. At 42-day old, twenty birds were selected based on average body weight of respective experimental unit (one bird each) and were decapitated and then were dissected to obtain intestinal digesta and meat samples. Meat samples were taken and analyzed for their protein content based on AOAC (1985) methods to determine the meat protein mass per 100 g of meat. The intestine was separated and cut according to its segments. The jejunal digesta sample was taken to calculate the populations of lactic acid bacteria (LAB) and coliform using the total plate count method as described by Barrow and Feltham (1993). While, the ileal digesta sample was taken to measure protein digestibility by the method of Lemme et al (2004). Similiar procedure was performed for caecal digesta sample to analyze the concentration of short-chain fatty acids using gas chromatography according to the method as described by Jensen et al (1995). After removing digesta, the length and weight of each segment of the intestine were measured. The jejunal segment was cut in 1 cm length, and put into 10% formaldehyde solution for futher villi length measurement using hematoxylin and eosin staining method as described by Nourmohammadi and Afzali (2013). Data were analyzed using analysis of variance and continued to orthogonal contrast test at 5% significant level.

Results and discussion

The jejunal lactic acid bacteria (LAB) and coliform counts (Table 2) in the broiler given diet composed of low level of intact protein (CP18) as well as microparticle protein with organic calsium (MCP18) added with additive did not different to those of control (CP21). These results indicated that the use of additives (Lactobacillus acidophilus or citric acid) in low protein diets (CP18 and MCP18) were able to modulate the development of intestinal microflora similar to high protein diets (CP21). This phenomenon was occured because both Lactobacillus acidophilus and citric acid have an important role in suppressing pathogenic bacterial growth, and finally maintain intestinal microflora balance. The colonization of Lactobacillus acidophilus on the intestinal lumen was able to compete with pathogenic bacteria. However, in case of citric acid supplementation brought about the decrease pathogenic growth due to the acid penetration into te body cell of bacteria.Therefore, the stable intestinal microflora was caused by the activity of Lactobacillus acidophilus and citric acid to suppress the development of coliform and encourage the development of beneficial bacteria. The improvement of intestinal microflora balance has been previously reported was due to the role of Lactobacillus acidophilus as probiotics (Jahromi et al 2016; Li et al 2018) and citric acid as an acidifier (Sultan et al 2015; Natsir et al 2017). Probiotic and acidifier have different mechanisms in improving the balance of intestinal microflora. Probiotics could improve intestinal microflora by several mechanisms, namely, inhibition of pathogenic bacterial colonization through competitions to obtain nutrients and attachment to the intestinal wall, the production of bactericidal compounds, and also due to neutralizing enterotoxins (Pan and Yu 2014). In case of the work of acidifier, it could directly penetrated bacterial cell walls and interfered with the normal physiology of harmfull bacteria that cannot tolerate wide range of intestinal pH gradients (Khan and Iqbal 2016). Therefore, the activity of both types of additive were able to maintain balance of intestinal microflora, especially in low protein diets, similar to normal protein fed birds.

The coliform population (Table 2) in the treatment of CP18 with addition of either Lactobacillus acidophilus (3.73 x 10⁷ cfu/g) or citric acid (2.58 x 10⁷ cfu/g) was higher (p <0.05) than that in the MCP18 treatment with similiar additive (4.67 x 10 ⁶ and 5.90 x 10⁶ cfu/g respectively). Thus, MCP18 with the addition of Lactobacillus acidophilus or citric acid had better ability to suppress coliform growth compared to CP18 which also received the same additives. The LAB population in the MCP18 with the addition of either Lactobacillus acidophilus (3.47 x 10 ¹⁰ cfu/g ) or citric acid (3.48 x 10¹⁰ cfu/g) was not different from that in the CP18 with the same additives (2.77 x 10¹⁰ and 1.52 x 10¹⁰ cfu/g, respectively). This phenomenon was closely correlated with the use of microparticle protein in the MCP18.

Microparticle protein have higher solubility (Hu et al 2013) because of their small particle size (O 'Sullivan et al 2016). Suthama and Wibawa (2018) reported that protein and amino acids digestibility and Ca retention increased in broilers fed with microparticle protein from fish meal and soybean meal in pellet form. High-utilization of microparticle protein is possibly supported the growth of intestinal lactic acid bacteria. The development of lactic acid bacteria in the intestine suppressed coliform growth resulting in a balance of intestinal microflora. The use of protein by lactic acid bacteria was in accordance to Apajalahti and Vienola (2016) who stated that the small intestine is dominated by LAB that has complex nutrient requirements. It has also been reported that Lactobacillus sp. (83% of the small intestinal bacterial population) could not synthesize amino acids and must be supplied from the availability of exogenous amino acids that derived from feed. Therefore, the role of microparticle proteins in the MCP18 is important in increasing the work of additives (Lactobacillus acidophilus and/or citric acid) to maintain the balance of intestinal microflora.

The present result also showed a change in short chain fatty acid (SCFA) in the 42-day old broiler cecum (Table 3). In general, the intact (CP18) and microparticle (MCP18) of low protein diets increased (p <0.05) propionic acid but did not affect cecal acetic and butyric acids compared to control (CP21). However, the increased SCFA was clearly indicated in broiler cecum due to feeding MCP18. The MCP18 had higher acetic, propionic and butyric acid (p <0.05) than the CP18 and control. The combination of MCP18 and Lactobacillus acidophilus or citric acid has been shown to increase SCFA production compared to other treatments. This phenomenon was supported by two factors, firstly, the stable balance of intestinal microflora (Table 2) and secondly, the increase in available substrates, especially soybean-oligosaccharides derived from microparticle soybean meal. Soybean meal contains large amounts of carbohydrates consisting mainly of non-starch polysaccharides and oligosaccharides (Choct et al 2010). Oligosaccharides and non-starch polysaccharide are substrates that can be fermented by lactic acid bacteria to produce SCFA (Scheppach 1994). Experimental diets composed of corn and soybean meal supported the development of LAB and production of SCFA in the digestive tract. The amount and type of substrates affect the composition of the digestive tract microflora and have an impact on the amount of SCFA production (Besten et al 2013). Citric acid supplementation in MCP18 increased (p <0.05) cecal propionic acid compared to feedingLactobacillus acidophilus (Tabel 3). Nevertheless, the addition of Lactobacillus acidophilus and citric acid in MCP18 is assumed to be suitable in increasing SCFA production.

Additives inclusion (Lactobacillus acidophilus or citric acid) in CP18 and MCP18 diets increased (p <0.05) the length of the colon (Table 4), the relative weight of the duodenum, jejunum and colon compared to CP21 (Table 5). This phenomenon indicated that the use of additives in low protein diets is able to stimulate the development of the digestive tract even though it got a lower protein intake than the treatment of higher protein diet (CP21). The use of additives created a balance of microflora in the digestive tract (Table 2) and then encouraged SCFA production (Table 3). Short chain fatty acids production resulted by the fermentation activity of lactic acid bacteria play a role in stimulating the development of the digestive tract. Previous research (Sohail et al 2013) showed the same phenomenon, that the administration of probiotic and prebiotic could increase the relative weight of the spleen, bursa, intestine and cecum in broilers with decrease in intake due to heat stress. Pan and Yu (2014) reinforced the phenomenon as mentioned above that SCFA has a role in stimulating the growth and proliferation of enterocytes, regulating mucin production, and influencing the intestinal immune response, thereby, encouraging intestinal development. Short chain fatty acids have the most impact on increasing length (Table 4) and relative weight of the colon (Table 5), due to the possibility of high absorption of SCFA in the colonic lumen. Absorption of SCFA in the colon reached to 95-99% of total results of bacterial fermentation (Scheppach 1994).

Feeding either intact (CP18) or microparticle (MCP18) protein diets at lower level with addition of additive (Lactobacillus acidophilus or citric acid) in broiler had no effect on the jejunal villi length, ileal protein digestibility (Table 6) and feed conversion ratio (Table 7) as compared to control. However, MCP18 treatment resulted in the increased ( p <0.05) meat protein mass (Table 6) and the decreased average daily feed intake, final body weight and daily body weight gain (Table 7) when compared to other treatments. This phenomenon showed that feeding Lactobacillus acidophilus or citric acid could improve gut health and increase the efficiency of protein utilitation. Giving Lactobacillus acidophilus or citric acid as a feed additive created a healthy intestinal condition by suppressing the growth of coliforms and supporting the development of beneficial bacteria (Table 2). Healthy intestinal condition encourage the development of intestinal villi and then increase protein absorption. This mechanism as described above resulted the better villi length and the protein digestibility in broiler fed low protein diet with addition of additive which was able to compensate the high protein diet (CP21). Previous studies (Incharoen et al 2010; Laudadio et al 2012) revealed that intestinal villi length decreased in broiler caused by low protein intake without any additive. This phenomenon occurred because the digestive tract tissue has a relatively high level of protein replacement and requires the supply of more protein to meet the needs of basal metabolism and the development of intestinal structures (Abbasi et al 2014).

The treatment of MCP18 has the most effect on the increased meat protein mass (MPM) and the decreased average daily feed intake (ADFI), final body weight and daily body weight gain. This was due to MCP18 diet composed of microparticle protein derived from fish meal and soybean meal, therefore, soybean oligosaccharide in soybean meal also changed in size to be smaller. Soybean oligosaccharide in MCP18 is known as potential prebiotic which is more easily fermented by digestive tract bacteria, such as LAB (Table 2), so that SCFA (acetic, propionic and butiric acids) production increased (Table 3). The increased short chain fatty acids, especially propionic acid, affect the meat protein mass because it plays a role in inhibiting lipogenesis, thereby, causing an increase in protein deposition. This phenomenon was in accordance with Morrison and Preston (2016) who stated that lipogenesis and cholesterogenesis are inhibited by propionic acid in rat. The study results reported by Suthama et al (2018) that broilers fed with soybean oligosaccharide from soybean meal extract could increase protein deposition and meat fat and cholesterol. The present result was in line with some previous studies regarding the use of prebiotics. The use of prebiotics as a substrate for beneficial bacteria, such as LAB, produced short chain fatty acids could maintain the balance of intestinal microflora in broiler (Perdinan et al 2019) and in indonesian local chicken (Purbarani et al 2019). The improvement of intestinal microflora balance can be correlated with the improved meat quality as indicated by the increase in meat protein mass (Abdurrahman et al 2016b), the decrease in meat fat mass (Abdurrahman et al 2016a) and meat cholesterol mass (Fajrih et al 2014; Wulandari et al 2018) in Indonesian crossbred local chicken.

The reduced ADFI, final body weight and daily body weight gain due to the increased SCFA production in MCP18 treatment (Table 7) with a mechanism as explained in the previous paragraph. Short chain fatty acids (especially acetic and propionic acids) that have been absorbed can be utilized to meet energy needs. Butyrate acts as the main energy source for the growth of large intestine, while propionate through the process of gluconeogenesis was mostly occurred in the liver, and the rest was transported to the brain, muscles, pancreas and adipose tissue as metabolic control and appetite regulators (Morrison and Preston 2016). The use of SCFA as an energy source can increase dietary energy metabolism. An increase in dietary energy metabolism leads to faster fulfillment of energy demand and resulting in a decrease in feed consumption. Previous studies showed the same results, that propionate is specifically absorbed and used as an energy source, so that excess of absorption can reduce feed intake in broiler (Pinchasov and Jensen 1989; Hume et al 1993; Pinchasov and Elmaliah 1994). The decreased feed intake was an effect of the high level of SCFA absorbed by the intestine was also reported by Byrne et al (2015). The decrease in ADFI in MCP18 treatment has an impact on lowering daily body weight gain and final body weight compared to other treatments. However, the feed conversion ratio in MCP18 was better than that in CP18 and almost equal to that of CP21. (texto entre tablas 6 y 7)

Conclusion

• Feeding diet composed of microparticle protein at low level and organic calcium with addition of Lactobacillus acidophilus or citric acid can stimulate health and development of broiler intestine.
  
  
• The addition of additive (Lactobacillus acidophilus or citric acid) in diet composed of microparticle protein at low level with organic calcium bring about an increase in short chain fatty acids production and leads to the decrease average daily feed intake, daily body weight gain and final body weight with the same feed conversion ratio, but increase meat quality indicated by meat protein mass.

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Received 12 February 2020; Accepted 5 March 2020; Published 1 April 2020

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