The ban on the use of antibiotic growth promoters (AGP) has posed a negative impact on the growth and health of broilers, thus threatening the sustainability of the broiler production. To support the sustainability of the broiler industry, the presence of an alternative to AGP is very much needed. Various AGP alternatives have been explored, one of which is functional feed ingredients. Functional feed is generally defined as a feed ingredient that have potential health effects other than basic nutrition [1]. Several active components in the functional feed are believed to have critical roles in promoting health and well-being of the host, including prebiotics and antioxidant compounds. Aside from the high energy content, unripe banana flour (UBF) has been reported to be rich in soluble fibre and resistant starch that can act as a prebiotic agent [2, 3]. This property is highly beneficial for the intestinal microbial balance of broiler chickens. The UBF also showed high antioxidant activity [3, 4], which may be beneficial in improving the physiological condition of broilers reared under intensive production system. Taking all these above facts into consideration, UBF has great potential to be developed into functional feed ingredients for broiler chickens during the post-antibiotic era.

UBF is generally prepared from green bananas that have been abandoned or discarded during the fruit selection and processing. Discarded green bananas are so plentiful in some banana-producing areas that if not utilized, they can pose environmental problems [5]. Unripe banana flour can also be obtained from various underconsumed banana cultivars that have low economic value [4]. As a tropical country, Indonesia is home to various cultivars of bananas [6]. Some banana cultivars have a high economic worth, whilst others possess a low economic value due to a lack of customer demand (underconsumed banana). In the previous study, we evaluated some banana cultivars in Indonesia, including Klutuk, Norowito, Raja Bandung, and Pisang Hijau [4]. It was shown that all unripe banana cultivars are high in energy content and can support the growth of Lactobacillus casei (lactic acid bacteria-based probiotics), suggesting that all banana cultivars possess prebiotic potential. Based on the 2,2-diphenylpicrylhydrazyl (DPPH) assay, it was further shown that the underconsumed banana cultivars of Klutuk and Norowito have high antioxidant activity [4]. Owing to these conditions, the unripe banana cultivars, particularly Klutuk and Norowito, may be employed as local and cheap functional feed ingredients for broilers.

Currently, the use of UBF for broiler chicken diets is still limited. To the best of our knowledge, only Dumorné et al. [7] used UBF as an energy source for broiler chickens. They confirmed that the inclusion of banana flour into broiler feeds of up to 20% did not adversely affect the growth performance of broilers [7]. However, the use of UBF as a functional feed ingredient and its impact on the physiological condition of broiler chickens have not been studied. Besides being applied individually, active components such as prebiotics are often combined with other active ingredients to obtain a synergistic effect so as to further increase their effectiveness in improving the physiological condition and health of broiler chickens. In this study, UBF as a functional feed ingredient was combined with probiotics or enzymes, and further evaluated its impact on physiological conditions in broilers. Overall, the purpose of this study was to determine the effect of UBF given individually or in combination with probiotics or enzymes on blood profile, serum biochemical parameters and intestinal indices of broiler chickens.

On day 21, the chicks in UBF group had relatively lower levels of MCV as compared to other treatment groups. In most conditions, the low MCV is generally attributed to anemia condition in chickens. However, the anemia seemed not to occur in our current study as the levels of MCV in all treatment groups were within the normal range (normal MCV values in broilers are 90-140 fl [11]). Also, the difference in MCV levels was no longer observed on day 38 across the treatment groups. On the measurement of day 38, the values of haemoglobin, MCH and MCHC were higher in UBFZYM than those in CONT and UBF chicks. A previous study by Attia et al. [12] reported that multienzymes supplementation was associated with the enhanced levels of haemoglobin, MCH and MCHC of broilers. Literature further suggested that multienzymes treatment improved the apparent nutrient digestibility of broilers, and thus enhanced the availability of nutrients for energy metabolism to support the growth [12, 13]. Considering the general role of haemoglobin in supporting the metabolic activity of chickens, the increased levels of haemoglobin, MCH and MCHC due to multienzymes supplementation may therefore be a response of broilers to supply more oxygen to cells for the metabolic activity of chickens. Our inference was actually supported by the fact that UBFZYM chickens had the highest final body weight among the groups of chickens. With regards to the absence of effect of unripe banana flour (source of prebiotics) and probiotics on the haemoglobin profile of broilers, the corresponding results were also reported by other authors in which administration of probiotics Lactobacillus paracasei [14] and Bacillus coagulans or Lactobacillus sp. [15]. resulted in no impact on erythrocyte profiles including haemoglobin, MCH and MCHC of broilers. Moreover, Al-Khalaifa et al. [15] documented no effect of prebiotics (mannan-oligosaccharide and fructo-oligosaccharide) on haemoglobin, MCH and MCHC of broilers.

It was shown in this present study that the numbers of leukocytes were higher in UBFZYM than that in CONT, while the effect of unripe banana flour was not substantial on the numbers of leukocytes. This may suggest that irrespective of the unripe banana flour effect, the multienzyme supplementation resulted in improved immune competencies of broilers. Our result was in accordance with Attia et al. [13] showing an increased number of leukocytes and lymphocytes with multienzyme+phytase supplementation in broiler diets. In line with this, Attia et al. [12] documented that enzyme supplementation improved immune competencies as indicated by the improvement in lymphoid organ development of broilers. They further confirmed that the increased follicular diameter of the bursa of Fabricius due to exogenous enzymes resulted in increased lymphocyte cell production. In accordance, the higher number of lymphocytes was also observed in the UBFZYM group as compared to other groups in this study. Attia et al. [12] further suggested that the improved immune system of enzyme-treated broilers was also due to the increased nutrient available for the production of immune cells.

It was clear in this present study that the levels of uric acid were lower in UBFZYM than in the control group. In most conditions, the increased blood uric acid levels may be attributed to muscle proteolysis (protein degradation) in poultry [16]. One of the factors that can induce protein degradation is low nutrient digestibility so that the availability of nutrients, especially protein, decreases. To address the lack of protein needed for physiological processes, the body will increase muscle proteolysis, as a result, uric acid production increases [16]. Considering the role of multienzyme in enhancing nutrient digestibility especially protein [12, 13], the low levels of uric acid in the serum of broilers treated with multienzyme may therefore be attributed to the less muscle protein degradation or sufficient protein supply in the body to support physiological processes of broilers. Our latter inference was actually supported by the highest final body weight of multienzyme-treated broilers as compared to other chicken groups. In this study, the effect of unripe banana flour was not substantial on the levels of uric acid. With regard to the prebiotic content of unripe banana flour, a study by Biswas et al. [17] noticed that prebiotic (fructo-oligosaccharides and mannan-oligosaccharides) treatment decreased uric acid concentration in the blood of broilers. In contrast, a study by Tang et al. [18] revealed that prebiotic treatment did not affect the levels of uric acid in laying hens. The differences in the nature and level of prebiotics administrated to poultry feed, feed composition (protein content), as well as the species and age of poultry were most likely to be responsible for the inconsistent impact of prebiotics on the protein metabolism and hence uric acid levels in the blood.

It was shown in this present study that on day 38 the pH values were lower in the duodenum of UBFPRO broilers as compared to other groups. Considering the absent difference in duodenal pH between CONT and UBF, the lower duodenal pH values of UBFPRO could be accounted to the role of probiotics administrated in the diets. This inference was supported by Machado et al. [19] who reported that the low pH values in the intestine of probiotic-treated chicks were attributed to the high population of lactic acid bacteria which produce lactic acid, and thus lowering the pH values of the intestine. In contrast to that of occurring in the duodenum, the pH values were higher in the caecum of UBFPRO as compared especially to control broilers. This finding was different from Machado et al. [19] showing the lowest pH values in the caecum of broiler receiving probiotics. In this study, the rationale for the higher caecal pH values due to probiotic treatment remains unclear. However, the improved nutrient digestibility and absorption in the upper intestinal part due to probiotics [20] may reduce the amount of feed reaching the caecum. The latter condition may therefore reduce the amount of substrates available for the growth of lactic acid producing bacteria and thus having an impact on the higher pH value in the cecum. With regard to the condition in ileum, the pH values were higher in UBFZYM than that in CONT broilers. In line with our finding, Gao et al. [21] reported that the pH of the digesta in the intestine (duodenum and jejunum) increased following the exogenous enzyme (xylanase) supplementation. Similar to the conditions in this present study, the elevated pH values in the ileum of broilers supplemented with multienzyme appeared to be related to more complete nutrient digestion (due to greater enzymatic activity of multienzyme), which therefore limited the amount of substrate available to support the growth of lactic acid bacteria in the ileum of broilers [19, 21].

It was shown in this study that the effect of unripe banana flour was not significant on the numbers of selected bacteria in the ileum and caecum of broilers at the measurements of day 21 and 38. Being considered as prebiotic source, the unripe banana flour neither increased the numbers of lactic acid bacteria, nor lowered coliform, lactose negative Enterobacteriaceae and Enterobacteriaceae. Previous study by Al-Khalaifa et al. [15] did not observe any influence of dietary administration of fructo-oligosaccharides (FOS) and mannan-oligosaccharides (MOS) on the numbers of lactic acid bacteria and Escherichia coli in the intestine of broilers. The inconsistent results of prebiotics on broiler chickens were also reported by Yang et al. [22]. They suggested that the inconsistent results when using prebiotics as feed additive may be attributed to the different natures and levels of prebiotics, species or strains of broilers and diets used during the study, as well as rearing conditions. In this study, the numbers of lactic acid bacteria and lactic acid bacteria to coliform ratio were higher in UBFPRO and UBFZYM than that of CONT. In respect of the lack effect of unripe banana flour, the administration of probiotics or multienzyme was therefore able to increase the growth of lactic acid bacteria in the ileum of broilers. Sugiharto [20] previously revealed that probiotics may create a more comfortable condition in the intestine (by producing organic acid and excluding potential pathogenic bacteria) and thus improve the growth of beneficial bacteria including lactic acid bacteria. In term of enzyme, Sugiharto [20] also suggested that exogenous enzyme may increase the production of lactic and other organic acids, which are very beneficial for the growth of lactic acid bacteria and other commensal bacteria in the intestine of broilers. The capability or enzyme in improving the availability of nutrients seemed also to be responsible for the availability of substrates needed by commensal bacteria to grow. The ratio of lactic acid bacteria to coliform bacteria in the intestine has been proposed as an indicator of gut health, with higher lactic acid bacteria to coliform ratio being associated with better intestine health in broilers (Vimon et al., 2020). Indeed, a higher lactic acid bacteria count is expected to inhibit the colonization of pathogenic bacteria such as coliform in broiler intestines [20, 23]. In line with the lactic acid bacteria population, broilers given probiotics or multienzyme had higher lactic acid bacteria to coliform ratio levels in their ileum than that of controls.

A study revealed that prebiotics improved the intestinal morphology of broilers [20]. In line with this, Alyileili et al. [24] found that using degraded date pits as a prebiotic source increased villus height, villus height to crypt depth ratio, and decreased crypt depth in broiler intestine. Although it was not significantly observed in the small intestinal segments at day 21 or the duodenum and jejunum at day 38, the ileum of UBF chicks had a higher villus height to crypt depth ratio than the control at day 38. This may therefore suggest that unripe banana flour can act as a prebiotic, which can help improve the morphology of broiler ileal segments. In poultry, a higher villous height to crypt depth ratio indicates a greater capacity for nutrient digestibility and absorption [25]. According to observations on the duodenal segment on day 38, the ratio of villous height to crypt depth in UBFZYM was significantly higher than in other birds. Given the importance of the duodenum in digestive and absorptive functions, the higher ratio of villous height to crypt depth may indicate that UBFZYM had better digestive and absorptive capacity than the birds in the other treatment groups. Indeed, the fact that the UBFZYM had the highest average body weight gain and feed efficiency among the bird groups supported our inference. In this present study, the higher ratio of villous height to crypt depth in the duodenum of UBFZYM broilers was attributed to the lower crypt depth in the respective chicks. Longer villi and shallower crypts, according to Alyileili et al. [24], promote nutrient absorption by activating intestinal cell maturation and digestive enzyme activity. In this regard, the lower crypt depth of UBFZYM may be attributed to the chicks’ improved nutrient absorption as well as a reduction in the metabolic cost of intestinal epithelium turnover [26]. In this study, the higher duodenal villus height to crypt depth ratio of the UBFZYM chicks appeared to be attributed to the role of enzymes in improving the morphology of the broiler small intestine. Previously, Yang et al. [27] confirmed that xylanase supplementation of wheat-based diets did not change villus height but did reduce jejunal crypt depth in 7-day-old broilers. Moreover, Mathlouthi et al. [28] found that 22-day-old broilers fed diets based on corn or wheat with β-glucanase + xylanase had higher villus height and villus height to crypt depth ratio in the ileum than those fed a wheat-based diet with no enzyme supplementation.

The current study found that using unripe banana flour improved broiler growth performance and feed efficiency of broiler chickens. These improvements could be attributed to the functional properties of unripe banana flour, such as prebiotic activity [4]. Indeed, research has shown that prebiotics can improve the digestive and absorptive functions of the broiler intestine [20], as evidenced by a higher villus height to crypt depth ratio, especially in the ileal segment in the present study. The antioxidative property of unripe banana flour [4] also appeared to be responsible for the improvement in physiological conditions, which ultimately increased energy allocation for growth. The combination of unripe banana flour and probiotic or multienzyme was expected to exert a synergistic effect on broiler growth performance. However, such a synergistic effect on broiler growth performance did not appear in the current study. Several factors, such as enzyme types and levels, prebiotic types, probiotic levels and strains, and so on, may influence the magnitudes of synergism among functional properties as revealed by El-Sayed et al. [29] (Table 1).

The broiler growth performance was improved when UBF was given alone or in combination with probiotics or multienzymes. The use of probiotics or multienzymes in combination with UBF improved intestinal bacterial population, while multienzymes improved broiler physiology and intestinal morphology.