Antibacterial activity of lactic acid bacteria from black soldier fly (Hermetia illucens) larvae fed with empty fruit bunch and tofu waste

Antibiotic Growth Promoters (AGP) are prohibited for use in livestock due to their detrimental effects on consumer safety and livestock productivity. Probiotics are an alternative to AGP because they offer microorganisms that are beneficial to the livestock health. In this investigation, Lactic acid bacteria (LAB) from the Black Soldier Fly (BSF) larvae’s will be isolated, tested for their antimicrobial activities and molecularly identified using 16S rRNA gene with Polymerase Chain Reaction (PCR) technique. The larvae are fed with empty fruit bunch and tofu wastes as a source of prebiotics to the indigenous LAB. The LAB isolates were enumerated and morpho-biochemically characterized. Disk diffusion method was used to determine the antibacterial efficacy of LAB isolates against pathogenic bacteria, such as Salmonella enterica serotype Pullorum, Salmonella enterica serotype Typhimurium ATCC® 14028, and Escherichia coli ATCC® 8739. Nine bacterial isolates were purified from a population density of log 6 Colony-forming unit (CFU)/mL on de Man Rogosa Sharp (MRS) agar. Four LAB isolates were confirmed through (-) catalase activity and acids production (homofermentative). Two LAB isolates namely 5P3i3 and 5P3i5 were designated as the most potential strains against three pathogenic bacteria with the strongest inhibitory activities against E. coli, followed by S. enterica ser. Typhimurium and S. enterica ser. Pullorum. By using 16S rRNA gene sequencing, the two LAB isolates were assigned to Lactiplantibacillus pentosus and submitted to GenBank as L. pentosus strain 5P3i3 (OP445834.1) and L. pentosus strain 5P3i5 (OP445835.1). The results of this study will be further investigated with the characterization of potential isolates as probiotic candidates to be supplemented in poultry.


Introduction
Antibiotics application in livestock has been explicitly prohibited under a National Law on Livestock and Animal Health No. 18 of 2009, Article 22, Paragraph 4c (UU No. 18 Tahun 2009 Pasal 22 ayat 4c), as decreed by the Indonesian Ministry of Agriculture.Apart from their role in disease prevention and treatment, antibiotics have long been employed in animal husbandry as antibiotic growth promoters (AGP), a practice dating back to the 1940s, aimed at enhancing the efficiency and productivity of animal feed [1].However, the application of antibiotics poses cryptic risks to human, including the deposit or accumulation of antibiotic residues in livestock meat and the introduction of antibiotic-resistant pathogenic bacteria [2].Therefore, there is an urgent need for antibiotic alternatives in order to optimize livestock productivity while ensuring the safety of consumable products, IOP Publishing doi:10.1088/1755-1315/1352/1/012011 2 preferably in the form of probiotics, thus mitigating the potential adverse consequences associated with antibiotic use [3].
Probiotics are live microorganisms that are administered in sufficient amounts and are primarily derived from the lactic acid bacteria (LAB) group which are beneficial for the health of the host [4].Lactic acid bacteria belong to a class of facultative anaerobic bacteria known for their ability to elevate intestinal acidity and inhibit the proliferation of pathogenic bacteria in various symbiotic environments, encompassing both animals and humans [5].This bacterial group exhibits the capacity to generate lactic acid, hydrogen peroxide, antimicrobial agents, and other bioactive substances that contribute to host well-being and productivity [6].The LAB category comprises several groups, including genera such as Leuconostoc, Lactobacillus, Streptococcus, and Pediococcus [7].Given that LAB populations dominate the intestinal microflora, it is feasible to isolate LAB from the digestive tract.Black soldier fly (Hermetia illucens) is an example of an animal that has a unique digestive tract and is likely to be colonized by strains and species of LAB which are potential as probiotics [8].
The presence of lactic acid bacteria (LAB) in H. illucens larvae has been demonstrated in numerous studies, employing various modifications in rearing conditions and dietary compositions.Altering the rearing temperature of H. illucens larvae, for instance, has revealed a prevalence of Enterobacteriaceae groups including Providencia at hotter temperatures [9].Osimani et al. [8], in their investigation of microbial populations in H. illucens fed with microalgae and coffee silverskin, reported a predominant count of viable enteric bacteria including Leuconostoc and Lactobacillus from the larval digestive tract.In a separate study, Campbell et al. [10] found that when H. illucens larvae were fed brewer's byproducts, Enterobacteriaceae and LAB were the dominant groups.Hermetia illucens larva exhibit the potential to convert and valorize agroindustry byproducts, livestock manure, urban solid waste, and other materials.Furthermore, they could serve as a source of potentially valuable LAB, warranting further exploration for probiotic applications [11].
In this investigation, we endeavored to rear H. illucens larvae using empty fruit bunches from oil palm and tofu waste as a food source.Subsequently, we assessed the presence of LAB in the harvested larvae.The outcomes of this study were to give a preliminary information to the understanding of H. illucens larval growth and the potential cross-application of LAB from insects to livestock.

Feeding substrates
The main component of the feeding substrates consisted of oil palm empty bunch, obtained from the chopping and washing process at a local plantation while the solid tofu wastes were collected from a local industry in Medan City.The feeding substrates (in a 1:1 ratio) were subsequently ground into particles approximately 0.5 mm in diameter using a food blender.These particles were then moistened with 1 liter of water.

Insect rearing
Six-day-old larvae, totaling 150 individuals, were placed in plastic containers containing 3 kg of the prepared feeding substrates.These containers were sealed with fine-mesh cotton gauze and maintained in a controlled environment at 28±2.0°C, with a relative humidity of 70%, in continuous darkness over a 24-hour period.Post a two-week period, the larvae were harvested to extract LAB.

Isolation and characterization of lactic acid bacteria from H. illucens larvae
Approximately 10 grams of randomly chosen H. illucens larvae (10 individuals) were ground using a mortar and pestle.Subsequently, 1 mL of the resulting slurry was transferred into 9 mL of sterile physiological saline and thoroughly mixed by vortexing.Ten-fold dilutions of these homogenates were then plated onto de Man Rogosa Sharpe Agar (MRSA) containing 1% CaCO3 (w/v).The culture plates were incubated at 37°C for 72 hours.The presence of lactic acid bacteria isolates was indicated by the development of a halo zone surrounding the colonies (Figure 1).Each colony was later purified and characterized using established procedures to identify LAB isolates exhibiting traits such as negative catalase activity, gram-positive staining, and the ability to produce acid (either homofermentative or heterofermentative).

Antibacterial test against pathogenic bacteria
Bioactivity of LAB isolates producing antibacterial compounds against pathogenic bacteria, including Salmonella enterica serotype Pullorum, Salmonella enterica serotype Typhimurium ATCC® 14028, and Escherichia coli ATCC® 8739, was evaluated using the disc diffusion method.Standardized inocula (approximately 10 8 CFU/mL) were prepared by suspending colonies from LAB isolates and indicator bacteria in physiological saline.Sterile cotton swabs were then immersed in each pathogen suspension and used to evenly spread the bacteria on Mueller Hinton Agar (MHA), after which they were allowed to air dry.Fifty microliters (µL) of LAB suspension were loaded onto disks, and ciprofloxacin served as the positive control, while sterile distilled water was used as the negative control.These disks were placed onto the surface of the MHA plates.The plates were subsequently incubated at 37°C for 24 hours, and the presence of a clear zone around the disks indicated the antimicrobial activity of LAB.The diameter of this zone was measured using a digital caliper in millimeters.

Identification of potential LAB using 16S rRNA
Genomic DNA isolation, PCR amplification (using primers 785F and 907R) and subsequent 16S rRNA sequencing were outsourced to Macrogen Inc., Singapore.Two prospective LAB isolates were accurately identified, and their corresponding sequences were submitted as records to GenBank.To ascertain the species, these sequences underwent a BLASTn analysis on the NCBI platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find the closest match.Subsequently, a phylogenetic tree was generated using MEGA11 to illustrate the species identity concerning other species included as ingroups [12].

Results and discussion
The typical characteristics of bacterial colony from H. illucens larvae were: round-shaped, smooth edge, flat to dome surface, and white to cream color (Table 1).Similar colony characteristics of isolates were also reported by Amelia et al. [5] regarding LAB isolates obtained from dadiah, a traditional fermented food from West Sumatra, Indonesia.The biochemical characteristics of LAB isolates are presented in Table 1.All LAB isolates were gram-positive with a dominant rod-shaped cell and catalase-negative.Only four isolates i.e 5P3i1, 5P3i2, 5P3i3, and 5P3i5 that were regarded as lactic acid bacteria based on their fermentation profiles or being homofermentatives and the absence of gas in durham tubes during the biochemical test [5].The four isolates were then subjected to further tests.
The results of the isolation of lactic acid bacteria were obtained at a density of log 6 CFU/mL which was slightly lower than a previous study (log 6.5 to 7.8 CFU/mL) by Osimani et al. [8] using H. illucens with different feeding substrates.The result may be influenced by the type and preparation of feeding substrates given to H. illucens larvae during rearing.In addition, the presence of LAB isolates in H. illucens may also be derived from the feeding substrates, which is in line with reports regarding the presence of indigenous microbes (aerobic, anaerobic, facultative anaerobic in oil palm empty fruit bunches [13] and proteolytic bacteria from tofu waste [14].Antagonistic activity from LAB isolates were tested against human and poultry pathogenic bacteria as shown in Figure 2. The results showed that two LAB isolates (5P3i1, 5P3i2) did not display any inhibition against tested bacteria.Only two isolates namely 5P3i3 and 5P3i5 which showed antibacterial activities with the largest inhibition zone against E. coli (>19 mm) so this pathogen is classified as the most sensitive during the test.However, the inhibitory activity is still far less than that of the antibiotic, ciprofloxacin.Based on the classification by Morales et al. [15], the inhibition activity by LAB isolates against tested bacteria is categorized as strong (>10-20 mm).Lactic acid bacteria are able to produce antibacterial metabolites such as alcoholic compounds, bacteriocins, and organic acids, with strong antagonistic activity against a wide range of microorganisms, including pathogenic and spoilage microorganisms [16].Organic acid secretion is meant to alter the pH of the environment, making it unfavorable for the growth of competitors, or in this case spoilage and pathogenic microorganisms.Because of the low pH in the environment, organic acids become liposoluble, allowing them to pass through the cell membrane and into the cytoplasm of pathogens thereby disrupting their physiological balance [17].Based on the BLAST analysis, the potential strains, 5P3i3 and 5P3i5 from H. illucens larvae had 99.54% and 99.61% similarity with Lactiplantibacillus pentosus strain BMOBR061, respectively.The identity of potential strains was further confirmed through the phylogenetic tree construction as presented in Figure 3.The tree showed the similar position of each strain next to the branch of L. pentosus (MN880129) at 73% which is considered as statistically acceptable.The relative positioning of these two strains in the phylogenetic tree is influenced by the nucleotide composition including the insertion of unique codons.Reports on the presence of Lactiplantibacillus pentosus (syn.Lactobacillus pentosus) as a symbiotic microorganism in insects has been reported along with other Lactobacillus group as a part of the gut microbiota of Hermetia illucens [18].When considering the presence of L. pentosus, Tajabadi et al. [19] reported that dominance of lactobacilli, including L. pentosus was found in the stomach of honeybee (Apis dorsata).

Conclusions
The use of feed derived from plantation and industrial waste, specifically empty oil palm fruit bunches and solid tofu waste, demonstrates its contribution as a prebiotic for the presence of lactic acid bacteria in the digestive tract of Hermetia illucens.The ability of LAB isolates to inhibit the growth of pathogenic bacteria paves the way to further investigate into their potential as probiotics for its application in livestock and poultry.Two isolates identified together as Lactiplantibacillus pentosus is also a new report for the species of LAB isolated from these larvae.

Figure 1 .
Figure 1.Colonies of presumptive LAB isolates showing a clear zone around them on MRSA.

Figure 2 .
Figure 2. Antibacterial activity of LAB isolates from H. illucens larvae fed with oil palm empty fruit bunch and tofu waste using disk diffusion method.SDW = sterile distilled water.

Figure 3 .
Figure 3. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of strain 5P3i3 and 5P3i5 (shown in bold) with closely related species.The tree was constructed using neighbor-joining method and kimura-2 parameter model with bootstrap value (BV%) at the nodes calculated from 1000× simulations.

Table 1 .
Morphological, biochemical and microcospical of isolated bacteria from H. illucens larvae fed with oil palm empty fruit bunch and tofu waste.