Studies on intestinal ecology and growth performance of Tegal ducks fed with Lactobacillus casei and porang (Amorphophallus oncophyllus) tuber extract

Istna Mangisah^1,2, Lilik Krismiyanto¹, Vitus Dwi Yunianto Budi Ismadi¹, Mulyono Mulyono¹, Bambang Sukamto¹, Fajar Wahyono¹ and Nyoman Suthama¹

¹ Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang 50275, Central Java, Indonesia
istnamangisah@yahoo.co.id
² Vocational School, Diponegoro University, Semarang, Central Java, Indonesia Central Java, Indonesia

Abstract

This research aimed to investigate the effect of a mixture of Lactobacillus casei and porang tuber extracts (LCP) on intestinal microbial population and ecology, immune organs and growth performance of Tegal duck. A total of 150 of 4-weeks-old male Tegal duck were allotted to 3 dietary treatments, i.e. LCP0: Basal feed; LCP1: basal feed + 1ml LCP/100 g feed, LCP2: basal feed + 2ml LCP/100 g feed. The observed variables included intestinal microbial numbers, weight of digestive organs, weight of lymphoid organ and growth performance.

The mixture of Lactobacillus casei and glucomannan from porang tuber extract improved intestinal ecology, increased the weight of the immune organ “bursa of Fabricius” and improved growth and feed conversion.

Keywords: additive, coliform, lactic acid bacteria, symbiotic, villi height

Introduction

Eggs and meat of ducks provide a relatively affordable and available animal protein for consumers. One of the native or indigenous duck breeds that developed in Indonesia as meat and egg producer is called Tegal duck. They can be marketed at 7 to 10 weeks old according to consumer needs, because the meat at that age has the best quality (Iriyanti et al 2018).

Raising Tegal ducks of either traditional or semi-intensive system and provided with a poor quality diet leads to low productivity (Mangisah et al 2009). Feeding additive in the form of a combination of probiotics and prebiotics is a possible effort that can be applied to improve duck production performance. Lactobacillus as a probiotic could stimulate microorganisms capable in modifying the gastrointestinal condition through the improvement the growth of nonpathogenic and gram positive bacteria. These bacteria are able to produce lactic acid and hydrogen peroxide that can impact on the suppression of the growth of intestinal pathogens and provide a favorable condition to increase the digestion and utilization of nutrients, and feed efficiency. Previous studies indicated that dietary addition of probiotic Lactobacillus sp. was effective in increasing lactic acid bacteria population and decreasing total Coliform in the intestine of broilers (Cholis et al 2018; Sugiharto 2016). The beneficial effect of Lactobacillus sp. combined with ginger and turmeric extracts was also reported to be effective in lowering Escherichia coli counts and in increasing lactic acid bacteria population and antioxidant status (GSH-Px and SOD) of broiler fed self-formulated diet (Risdianto et al 2019). It was likely that Lactobacillus sp. can exert its positive impact on host animals with several states of diet condition. As it has been previously stated by Saputra et al (2020) that inclusion of Lactobacillus acidophilus in broiler diet composed of low level (18%) protein enhanced total lactic acid bacteria and depressed coliform counts due to the increased production of short chain fatty acid.

Lactic acid bacteria (LAB) require a source of "feed" in the form of prebiotics that can be fermented to produce lactic acid and short chain fatty acids (SCFA). These metabolites or products bring about the change in gut condition profile with reference to the balance improvement of intestinal microflora. This physiological phenomenon is closely related to the healthier environment of the digestive tract. One potential prebiotic that is widely used as “feed source” for Lactobacillus is known as glucomannan. Glucomannan can be found from “porang” tuber flour ( Amorphophallus oncophlus) at a level of 41% (Ngatirah and Syaflan 2016). The extraction of porang powder by maceration and ethanol leaching procedure produced the highest glucomannan and the lowest oxalate levels (Wijanarko, 2011).

Previous studies indicated that supplementation of probiotics and prebiotics can improve the performance of broilers (Salim et al 2013; Bozkurt et al 2014; Yuanita et al 2019; Setyaningrum et al 2019). Diet added with glucomannan alone extracted from porang tuber at 1.2% increased lactic acid bacteria population, decreased total Coliform, and improved body resistance of broiler chickens indicated by lower H/L ratio (Perdinan et al 2019). To date, very few studies have been comprehensively carried out to investigate the effect of feeding the mixture of Lactobacillus casei and porang tuber extract in poultry. For this reason, the current study was conducted to evaluate the effect of dietary inclusion of Lactobacillus casei and porang tuber extract mixture on intestinal bacteria population, weights of immune and digestive organs, intestinal villi height, and growth performance of male Tegal ducks.

Materials and methods

Ethical approval

The current study was carried out as a part of the Institute Research Project and the protocol was based on the rule of animal treating as appointed in the Republic of Indonesia’s law number 41, 2014.

Preparation of a mixture of LCP

The study was started with the extraction of porang tuber powder to obtain glucomannan based on the slightly modified method of Setyawati et al (2017). Porang tubers were harvested at ten month old, cleaned, sliced, dried and ground. Porang tuber powder (50g) was put in beaker glass containing distilled water with a ratio of 1:30 (w/v), and then was stirred using a homogenizer at 700 rpm for 15 minutes.

The mixture was continuously stirred at the temperature of 95ºC for 120 minutes. The mixture formed was centrifuged at 3000 rpm for 15 minutes to get the filtrate. The filtrate containing component of glucomannan was separated by filter paper. The precipatates of glucomannan were washed with 95% ethanol and were oven dried at 40 ºC for 24 hours. The dried glucomannan was ready to be used for LCP preparation, thereafter.

LCP was prepared by mixing Lactobacillus casei (10⁹ cfu/g) in 50 ml of 10% skim milk (10 g skim milk in 100 ml of sterile distilled water) and was incubated for 2 x 24 hours at 37^oC. Then, 50 ml of liquid mixture containing Lactobacillus casei was added with 2% (w/v) of sterilized porang tuber extract and was re-incubated for 2 x 24 hours to produce LCP. The bacterial population in LCP was counted with total plate count (TPC) method (Usman et al 2018).

Experimental animals and feeds

One hundred and fifty male Tegal ducks (age 30 days, with an average weight of 631±26 g were obtained from Livestock Breeding Center, Livestock Service Office, Central Java Province, Indonesia. The experiment was arranged in a completely randomized design comprising 3 treatments with 5 replications, and 10 ducks per replication. Treatments were addition of LCP extract (LCP0, LCP1 and LCP2) at 0, 1 and 2ml/g of feed (fresh matter basis). The basal diet was formulated according to the requirements recommended by Indonesian National Standards (SNI) SNI 8508:2018. The ration was prepared using maize, rice bran, soybean meal, meat and bone meal and premix (Table 1). The feed mixture was analyzed for its proximate composition using the AOAC methods (2007).

The ducks were adapted to the experimental diets from 15 to 30 days old and measurements made from 31 to 58 days old. Feed and water were given ad libitum.

Parameters measured

Gastrointestinal tract and immune organs

At the end of treatment, the ducks were were fasted for 15 hours then slaughtered by cutting their jugular veins. The small intestine (duodenum, jejunum, and ileum), caecum and colon were excised and weighed. Immune organs (spleen, thymus and bursa of fabricius) were removed and weighed.

Lactic acid bacteria and coliforms in the small intestine

Digesta (for pH, LAB and coliform determinations) was collected from the slaughtered ducks. pH values of intestine were determined according to Nabizadeh (2012). One gram of fresh digesta sample diluted into10 ml of distilled water was measured using a pH meter “smart Sensor pH 818”. Microbial population in the cecum (lactic acid bacteria and coliforms) was measured based on the method of Peng et al (2016), in which one gram digesta was transferred to 9 ml of sterile physiological saline solution, homogenized and diluted up to 10^-8. The suspensions were plated in de Man Rogosa and Sharpe Agar (MRSA) and incubated anaerobically at 37ºC for 48 h for the enumeration of lactic acid bacteria. For the enumeration of coliform, the homogenized samples were diluted up to 10^-6, then plated in McConkey Agar and incubated anaerobically at 37 ºC for 24 h.

Villi height of intestine

Observations of villi height (VH) were according to Setyaningrum et al (2019) with some modification. Specimens of intestine were cut around 2 cm, fixed in a 10% neutral buffer formalin solution for 2 days, dehydrated and then embedded in paraffin. The sections were cut at 4 µm and stained with haematoxylin and eosin. Villus height were measured under Leica ICC50HD microscope (4x magnification) with Leica Application Suite version 3.4.0 software. The height of the villi was measured from the end of villi to the intersection between villi - crypt.

Growth performance

Final (live) body weight was recorded at the end of the study after weighing weekly. Feed conversion ratio was calculated from the average of daily feed consumption divided by daily body weight gain

Statistical analysis

Treatment means were subjected to analysis of variance and the difference of means value was compared using SAS software based on Duncan’s procedure test.

Results

Gastrointestinal tract

Villi height was increased by LCP1 treatment but not by LCP2 (Table 2). The weight of ileum was increased with a curvilinear trend as LCP level was increased (Figure 1). The relative weights of duodenum, jejunum, caecum and colon were not affected by LCP supplementation.

Intestinal microbes

LCP increased LAB population, decreased Coliforms and decreased the pH of the intestine (Table 3).

Immune organs

LCP increased the weights of thymus, bursa of fabricius (Figure 2) and spleen (Table 4).

Figure 1. Effect of LCP supplementation on villi height	Figure 2. Effect of LCP supplementation on weight of bursa of Fabricius, % of slaughter live weight

Growth performance

The growth response to LCP was curvilinear increasing by 20% with addition of 1ml LCP/100g of diet, but with a lower rate of 15% improvement when the LCP was fed at 2ml/100g of diet (Table 5; Figure 3).

Figure 3. Effect of a dietary supplement of LCP on live weight gain of ducks	Figure 4. Effect of a dietary supplement of LCP on feed conversion of ducks

Discussion

Feed containing Lactobacillus casei and glucomannan from porang tuber extract (LCP) did not affect relative weight of duodenum, jejunum, caecum and colon (Table 2). This result is in accordance with Iriyanti et al (2018), that the intestinal profile weights percentage of intestine, gizzard, crop, and pancreas of ducks was not affected by prebiotic supplementation in feed. However, LCP supplementation increased the relative weight of the ileum. The increased ileum weight can be assumed that the condition much healthier than other segments due to the less pathogenic bacterial counts and higher growth of beneficial bacteria (LAB) in general (Table 3), although bacterial populations in the ileum did not specifically measured in the present study. It can be compared to the previous study that ileal LAB population decreased and E.coli counts decreased dramatically in crossbred local chickens fed prebiotic dahlia inulin (Krismiyanto et al 2014). Thus, this condition was possibly related to the change in microflora in the intestine leading to intestinal villi growth alteration (Table 3. Figure 1).

Dietary supplementation of Lactobacillus casei and glucomannan from porang tuber extract (LCP) provided beneficial effects on intestinal ecology and immune response in Tegal ducks. The change in intestinal ecology due to beneficial effect of supplementation of Lactobacillus casei and glucomannan from porang tuber extract mixture indicated by the increased total lactic acid bacteria and decreased coliform counts in the intestine (Table 3). Porang (Amorphophallus oncophyllus) tuber extract contains the same beneficial carbohydrate component as found in Amorphophallus konjac (KGM) known as glucomannan. Glucomannan with its carbohydrate component can be function as “source of feed/nutrition” for Lactobacillus casei in particular, and lactic acids bacteria in general. The presence of glucomannan can be fermented by Lactobacillus casei to produce short chain fatty acids (SCFA) which brought about the growth inhibition of pathogenic (coliform) and increased LAB population (Table 3). The present results were consistent with the previous reports in broiler fed a combination of Lactobacillus sp. and dahlia inulin (Cholis et al 2018), and inulin alone from dahlia tuber extract (Suthama et al 2019) that the intestinal bacteria changed to be the better balance with lower coliform and higher LAB populations.

There was not much known and was not specifically observed in the present study about the mode of action of mannose derived from MOS or glucomannan in reducing pathogens. However, it was reported that pathogens adhere to MOS particle in the form of mannose and are discarded (Sohail et al 2012; Spring et al 2000). Therefore, it can be briefly concluded that LCP supplementation can increase LAB population through the mechanisms of the two synergistic effects, Lactobacillus casei and glucomannan. The fermentation activity of Lactobacillus casei on glucomannan to produce SCFA that decreased coliform counts and increased LAB growth, was the indication of synergistic works of both. This result was supported by Perdinan et al (2019) who reported that dietary supplementation of glucomannan extracted from Amorphophallus oncophyllus tuber increased the BAL population, and decreased the number of coliform in the jejunum and ileum of broilers.

The results of study demonstrated a positive impact of feeding LCP that can increase intestinal villi height (Table 2). Villi height is an indication of greater surface area which in turn can increase nutrient digestibility and absorption capacity, and to a certain extent of intestinal health improvement. It was in accordance with the investigation of Purbarani et al (2019) that the increase in villi height induces the improvement of protein digestibility in crossbred local chicken fed diet with inclusion of a combination of Lactobacillus sp. and dahlia tuber inulin. Consistent with the present results, a previous study on feeding effect of inulin from gembili tuber and Lactobacillus plantarum demonstrated the increased villi height and the ratio of villi height to crypt depth in the duodenum, jejunum and ileum of broiler (Setyaningrum et al 2019). An increased intestinal villi height was associated with the changes in intestinal microbes due to dietary LCP supplementation. Intestinal microbial balance has a good effect on the growth of villi because LAB increased mucin production which protects intestinal epithelial cells from the toxins intervention that produced by pathogenic bacteria. This result was supported by the finding of Shirani et al (2019) that the increased microbial balance in GIT was an impact of intestinal pathogenic bacteria reduction, which caused intestinal structure and function improvement. The present study results are in line with Wang et al (2017) who found that supplementation of Lactobacillus plantarum increased villi height, villi height to depth of crypt in the jejunum.

In connection with the relative weights of the immune organs (spleen, bursa of fabricius and thymus), there was a significant increase in LCP treated groups as compared to control/LCP0 (Table 4; Figure 2). These results accordance with the finding of Willis et al (2007) and  Alkhaft et al (2010) that feeding probiotics in broilers increased the relative weight of spleen. The increase in the relative weight of bursa can be associated with the enhancement of immune cells number. The results found in this study were in agreement with Khan et al (2013) who found that bursa fabricius in the birds given probiotics showed an enhancement of the number of immune organ follicles. In relation to the weight changes in immune organs, it can be known that both bursa of fabricius and spleen weights increased (Table 4). The increased weight of spleen due to feeding effect of LCP can be still categorized in a state of good health condition. In case of the present study, the increased weight of spleen did not interfere with the health status since its work can be balanced by the role of bursa of fabricius. Thus, these results implied the positive feeding effect of Lactobacillus casei and glucomannan from porang tuber extract on the immune system improvement of Tegal duck.

The better villi growth indicated by villi height (Table 2), the greateris the  surface area for nutrient digestibility and absorption. The higher nutrient supply is an important key for growth performance improvement. The improvement of intestinal ecology and function of immune organs brought about the increased in final body weight and body weight gain (Table 5; Figure 3) with improved feed conversion (Table 5; Figure  4). This phenomenon was supported by the previous finding (Purbarani et al 2019) that feeding Lactobacillus sp. combined with dahlia tuber inulin improved villi height and protein digestibility, and finally brings about the increased carcass yield and final body weight. The lower level of LCP is considered to be the more appropriate since the growth improvement tended to decrease with the highest level of LCP (Figure 3).

Conclusion

• Feeding Lactobacillus casei combined with glucomannan derived from porang tuber extract led to: an increase in lactic acid bacteria (LAB), a decrease iintestinal pH and coliform numbers, and improved growth performance in Tegal ducks.
  
  
• Feeding the combination of Lactobacillus casei and glucomannan derived from porang tuber extract can be categorized as optimal at the level of 1ml/100 g feed.

Acknowledgments

This research project was funded by Ministry of Research, Technology and Higher Education, Republic of Indonesia through the Basic Research of Higher Education  , with contract No. 257-46/UN7.P4.3/PP/2019.

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