Abstract: Objective: This study aimed to analyze the effect of nano-encapsulation of noni (Morinda citrifolia) fruit extract in drinking water on antibacterial activity and the morphology of intestinal villi in laying hens. Materials and Methods: The experiment was conducted in a completely randomized design consisting of 6 treatments and 5 replicates, with 12 chickens (20 weeks) in each replicate pen. The treatments were: P0 = Water without an additive (negative control), P1 = Water+50 mg/hen of tetracycline (positive control), P2 = Water+0.5% extract of noni fruit, P3 = Water+0.5% nano-encapsulation of noni fruit extract, P4 = Water+1% nano-encapsulation of noni fruit extract and P5 = Water+1.5% nano-encapsulation of noni fruit extract. At the end of the experiment (5 weeks), parameters included bacterial populations in the small intestine (Escherichia coli, Bacillus subtilis and Salmonella sp.) and the morphology of intestinal villi (villus height, villus width, crypt depth and villus height: Crypt depth) were observed. Data were analyzed statistically using one-way ANOVA. Orthogonal contrasts were used to analyze all data with significant differences. Results: The results showed that the addition of 1% nano-encapsulation of noni fruit extract in drinking water did not affect the morphology of the intestinal villi or the populations of Bacillus subtilis and Salmonella sp., but it reduced the population of Escherichia coli (p<0.05). Conclusion: Supplementation with nano-encapsulation of noni fruit extract in the drinking water of laying hens reduced colonization by Escherichia coli and might be used as an alternative to antibiotics in laying hens.
INTRODUCTION
The presence of pathogenic bacteria in the digestive tract of laying hens decreases the quality of eggs and harms consumers1. The use of antibiotics to control pathogenic bacteria in laying hens has resulted in residues that can be found in eggs up to several weeks after administration2. One of the potential replacements for antibiotics is noni fruit (Morinda citrifolia). Noni fruit reportedly has some antibacterial compounds, including anthraquinones, alizarin, aucubin, saponin, L-asperuloside and scopoletin3,4. However, bio-active compounds such as polyphenols have low bioavailability. Polyphenol compounds are highly sensitive to gastrointestinal conditions (alkaline pH, enzymes and the presence of other nutrients) and are difficult to dissolve in water, making them difficult to absorb5. One of the efforts that can be taken to maintain the activity of compounds with low bioavailability is the nano-encapsulation method6. The advantages of this method are that particle size can be manipulated, a drug can be carried to the target organ, the activity of bio-active compounds can be maintained and the bio-active compounds can be absorbed faster7,8. Therefore, this study was conducted to observe the critical functions of nano-encapsulation of noni fruit extract, namely, the reduction of colonization by harmful intestinal microbes and the effect on jejunal morphology in laying hens.
MATERIALS AND METHODS
A total of 360 female ISA Brown chickens (20 weeks of age) obtained from a local company in Kediri, Indonesia, were used in the current study. Noni fruits were obtained from the Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia. Chitosan and sodium tripolyphosphate (STPP) were purchased from Bratachem, Yogyakarta, Indonesia. The experiment was conducted in a completely randomized design consisting of 6 treatments and 5 replicates, with 12 chickens in each replicate pen. The treatments were as follows: P0 = Drinking water without an additive (negative control), P1 = Drinking water+50 mg/hen of tetracycline (positive control), P2 = Drinking water+0.5% noni fruit extract (NFE), P3 = Drinking water+0.5% nano-encapsulation of NFE, P4 = Drinking water+1% nano-encapsulation of NFE and P5 = Drinking water+1.5% nano-encapsulation of NFE. The observed parameters were the microbial populations of the jejunal intestine, including Bacillus subtilis, E. coli and Salmonella sp. and the histomorphology of the jejunal intestine, including villus height, villus width, crypt depth and villus height: Crypt depth.
Preparation of noni fruit extract: A total of 1.5 kg of fresh noni fruits was blended and mixed thoroughly with 1900 mL of aqua dest for 15 min. The mixture was then filtered using nylon fabric. The liquid extract was heated at 90°C for 4 h to reduce the water content to form a paste. The paste was mixed with 575 mL of 96% alcohol using a magnetic stirrer (C-MAG HS 7, IKA, Selangor, Malaysia) for 20 min. Sediment precipitated from the solution after 20 min, at which point the solution was ready for nano-encapsulation.
Nano-encapsulation of noni fruit extract: Nano-encapsulation procedures were modified from Sundari et al.9. A total of 3.816 g of chitosan was dissolved in 610 mL of 2.5% acetate using a magnetic stirrer for 30 min. The chitosan solution was combined with 382 mL of noni fruit extract and stirred for 20 min. In a separate container, 0.125 g of STPP was dissolved in 16.7 mL of aqua dest. The STPP solution was then added to the chitosan-noni fruit extract solution and the mixture was homogenized using a magnetic stirrer for 20 min. The resulting solution was heated at 70°C for 1 h to evaporate the alcohol.
Populations of microbial intestinal pathogens
Bacillus subtilis: Jejunal digesta were planted in brain heart infusion agar (BHIA) medium according to Koshikawa et al.10. The sample was then incubated upside down at 37°C for 24-48 h. The number of bacteria was determined by the total plate count (TPC) method.
Escherichia coli: Eosin methylene blue agar (EMBA) medium was used according to Leininger et al.11. Jejunal digesta were inoculated on EMBA by the spread method. After being inoculated, the sample was stored upside down in an incubator overnight at a temperature of 37°C. The number of E. coli was calculated as a total plate count (TPC) by counting greenish colonies with black spots in the middle.
Salmonella sp.: One gram of jejunal digesta was inserted into nutrient broth and incubated for 24 h at 37°C and then planted on Salmonella shigella agar (SSA) medium according to Al-Lahham et al.12. Jejunal digesta were inoculated on SSA by the spread method and then incubated for 24 h at 37°C. The number of E. coli was calculated by the total plate count (TPC) method13.
Histomorphology of jejunal villi: The samples used for histomorphology were taken a 6 cm section of the jejunum. The jejunal digesta were removed and the intestinal mucosa was cleaned by physiological sodium chloride (0.89% NaCl in sterile water) and then stored in a 10% formalin buffer solution. The sample was then dehydrated in a series of acetone concentrations (35, 50, 70 and 95%)14 and embedded in paraffin. Villi tissues were then cut into 4 μm slices using a microtome and placed on slides to be stained by the hematoxylin-eosin method15. The observations of histomorphology of jejunal villi, including villus height, villus width, crypt depth and villus height: Crypt depth, were conducted using a light microscope with 4X magnification equipped with an Optilab digital camera (Optilab Advance, Miconos, Yogyakarta, Indonesia).
Statistical analysis: Microbial populations and histomorphology of jejunal intestine were compared between groups following a completely randomized design in a one-way arrangement16. The statistical analysis was conducted in Statistical Package for the Social Sciences (SPSS for Windows Version 16, SPSS GmbH, Munich, Germany). All significant differences were tested further using orthogonal contrasts. Indications of apparent differences in this study were based on a probability of less than 5%.
RESULTS AND DISCUSSION
The basal diet composition and nutrient content are represented in Table 1 and the data for bacterial populations in the jejunum are presented in Table 2. There was a significant effect (p<0.05) of supplementing with nano-encapsulation of NFE on the population of E. coli in laying hens. Supplementation with 1% nano-encapsulation of NFE in the drinking water of laying hens reduced the population of E. coli in the jejunum. The results of this study were in line with those of Kurniawan et al.17, who reported that supplementation with noni fruit reduced the amount of E. coli bacteria in the digestive tract of ducks. The presence of antibacterial compounds in noni fruit was thought to be the cause of the decline in E. coli populations. Saponins could change the permeability of microbial cell walls18. Aucubin inhibited the synthesis of RNA and microbial proteins and decreased the production of IL-6 and TNF in mast cells19. Flavonoids had an inhibitory effect on nucleic acid synthesis and energy metabolism and destructed the cytoplasmic membrane of pathogenic bacteria20.
The effects of supplementation with nano-encapsulation of NFE on the population of B. subtilis are summarized in Table 2. Initially, there was no significant effect (p>0.05) of treatment on the population of B. subtilis in the laying hens. The low population size of B. subtilis in all groups might be due to the sensitivity of B. subtilis to all treatments, including the controls. P0 had the largest E. coli population of all the treatments, so competition would occur between E. coli and B. subtilis for attachment to the intestinal wall21. The use of tetracycline in group P1 might have suppressed the growth of B. subtilis22. Tetracycline inhibits bacterial protein synthesis at the translation stage on the ribosome23. The presence of phenolic compounds in noni fruit could also have inhibited colonization by B. subtilis in groups P2, P3 and P4. Phenolic compounds have small molecules that penetrate the cell wall24. The Salmonella sp., bacterial population was not detected in any treatment. This might be caused by the effective application of a biosecurity program, such that Salmonella sp. was not able to grow in the digestive tract of laying hens. Good biosecurity programs such as maintaining the cleanliness of the cage and burying dead, infected chickens reduced Salmonella infections25.
The effects of supplementation with nano-encapsulation of NFE on villus characteristics are summarized in Table 3. Initially, there was no significant effect (p>0.05) of treatment on the villus characteristics of the jejunum, including villus height, villus width, crypt depth and villus height: Crypt depth. This result differed from that of Kurniawan et al.17, who reported that supplementing feed with noni fruit could increase villus height. This difference in results is thought to be due to a difference in the types of livestock, since the study of Kurniawan et al.17 used ducks. The results of this study were also different from the results of the study by Sunder et al.26, which showed that noni fruit juice could increase the height of intestinal villi in the duodenum.
| Table 1: | Basal diet composition and nutrient content |
aVitamins and minerals premix content per kg: Calcium 32.5%, Phosphorus: 1.0%, Iron: 6 g, Manganese: 4 g, Iodine: 0.075 g, Copper: 0.3 g, Zinc: 3.75 g, Vitamin B12: 0.5 mg, Vitamin D3: 50,000 IU, bConversion values from 70% gross energy | |
| Table 2: | Microbial populations in the jejunum of laying hens given drinking water with nano-encapsulation of noni fruit extract for 35 days |
| Ud: Undetected, SEM: Pooled standard error of means from column-wise comparisons | |
| Table 3: | Jejunal morphology responses to administration of nano-encapsulation of noni fruit extract via drinking water in laying hens for 35 days |
VH: Villus height, VW: Villus width, CD: Crypt depth, V:C ratio: Villus height: crypt depth, SEM: Pooled standard error of means from column-wise comparisons | |
Supplementation with 1% nano-encapsulation of noni fruit extract in drinking water reduced the population of E. coli but did not affect the population of Salmonella sp. or B. subtilis, nor did it affect the histomorphology of the jejunal intestine. The current study showed a beneficial function of the nano-encapsulation technique, namely, to increase the efficacy of phytobiotics administration through drinking water to reduce populations of potentially pathogenic microbes. The reduction of harmful microbes may help to maximize nutrient uptake for daily body requirements.
CONCLUSION
Supplementation with nano-encapsulation of noni fruit extract in the drinking water of laying hens reduced colonization by E. coli and might be used as an alternative to in-feed antibiotics in laying hens.
SIGNIFICANCE STATEMENTS
This study discovered that supplementation with nano-encapsulation of noni fruit extract in the drinking water of laying hens can be beneficial by reducing colonization by E. coli. This study will help researchers to explore the use of nano-encapsulation for protection using plant extracts, especially from noni fruit. Therefore, a new theory may develop regarding the efficacy of nano-encapsulation of noni fruit extract.