The effect of alginate concentration on the viability of probiotics ( Bacillus subtilis ) in synbiotic microcapsule beads after incubation at 70°C

. High temperatures in feed molding machines can cause a decline in probiotic populations in fish feed. Adding alginate in probiotic preparations is expected to wrap probiotics so that when added to the feed before pelleting, it does not experience a drastic decrease. This study investigated the decline in probiotic populations in fish feed caused by high temperatures in feed molding machines and evaluated the use of alginate in probiotic preparations to prevent drastic decreases in viability. The study aimed to determine the viability of Bacillus sp. in synbiotic microcapsule beads with varying levels of alginate concentration after exposure to 70°C. The research used four treatments and three replicates with a randomized complete design. The results showed that the alginate concentration affected the diameter of the beads of synbiotic microcapsules, but it did not significantly affect the viability of Bacillus sp. The viability of probiotics in alginate beads after exposure to 70°C was 5.182±0.527 - 5.720±0.072 log cfu/ml. Therefore, using alginate in probiotic preparations can be considered as a reference for adding probiotics to fish feed before pelleting to maintain the viability of the probiotics in high-temperature conditions.


Introduction
The use of probiotics in various sectors has become common, as has the use of probiotics in aquaculture.Probiotics are live microorganisms that provide health benefits to their host when administered in the right amounts.In addition to improving the water quality of cultivation media, using probiotics in aquaculture is also expected to improve the growth performance of cultivation [1][2][3].This is possible because some probiotic isolates can produce enzymes that help cultivate and absorb food, improve intestinal bacterial communities, suppress pathogenic bacteria growth, and increase cultivation immunity [4,5].Some types of probiotics that are often used in aquaculture are Bacillus spp [6]; Bacillus licheniformis and Bacillus amyloliquefaciens [3]; Bacillus licheniformis, Brevibacillus, and Bacillus subtilis [7][8][9]; Lactococcus [10,11].In general, these probiotic preparations are in liquid/free-cell form.However, with the development of probiotic presentation to facilitate application and protect probiotic cells from environmental stress, probiotics have also been preserved in powder/powder [12,13] One method that can be used to protect probiotics is encapsulation.Encapsulation is wrapping (coating) a core material, in this case, microscopic organisms of probiotics as core ingredients, using certain beneficial ingredients to maintain feasibility and keep probiotics from extreme environmental conditions [14].The encapsulation method can be done with various techniques, including the extrusion method.The material that is widely used as a coating in the extrusion method is alginate.Alginate is an acidic polysaccharide derived from the cell wall of brown seaweed and can be found as an amorphous component of the cell wall.Alginate is a natural polymer with high molecular weight [15].This alginate coating will protect probiotics from harmful environmental conditions [16].Previous studies have also shown that microencapsulation of Bacillus sp using alginate can improve growth performance [17].
Based on the description above, this study aims to determine the viability of probiotics in synbiotic microcapsule beads using several levels of alginate concentration after exposure to a temperature of 70 o C for 60 minutes.Knowing the best probiotic viability can be a reference in mixing probiotics into feed formulations before pelleting, as it is known that the temperature in the feed molding machine tends to be hot and can interfere with the viability of probiotics when mixed without coating first.

Research methods
This research was conducted at the Health and Environmental Pathology Laboratory, Research Center for Brackish Water Aquaculture and Fisheries Extension (RIBAFE) Maros Regency, South Sulawesi.

Research procedure manufacture of synbiotic microcapsules
This study's procedure for making synbiotic microcapsules began with rejuvenating probiotic isolates [18].The probiotic used in this study was Bacillus subtilis isolated from the intestines of Rabbitfish Siganus guttatus.The streak method rejuvenated probiotic isolates on Tryptic Soy Agar plate media.This probiotic is then incubated for 48 hours at room temperature.After incubation, probiotics were harvested and propagated again in 100mL of Nutrient Broth (NB) media and then set on a shaker incubator for 24 hours, then sampled to determine the density of probiotics propagated.
The next stage is to centrifuge probiotics propagated in NB media.The condition of the centrifuge is a speed of 6000rpm for 10 minutes.The precipitate formed is then rinsed using a physiological solution of as much as 1x.The pellet is rinsed, and the re-centrifuged probiotic pellet is then put into 25 ml of sterilized 1% inulin solution.The probiotics in inulin are then incubated for 20 minutes on a shaker incubator and then put into 75 ml of alginate solution with alginate doses according to treatment (1, 2, and 3%).The probiotics in this alginate solution (synbiotic) are incubated again for another 20 minutes on a hotplate stirrer.After incubation, this synbiotic is printed using a 24 G syringe and dropped/dripped into a calcium chloride solution (0.45M CaCl2).The microcapsule beads formed are then filtered and stored in each container according to treatment.Shapes and diameters are documented and measured using microscopes equipped with cameras and their applications.

Incubation of synbiotic microcapsules at 70 o C
The synbiotic microcapsule beads were weighed 1 g each and then put into the Erlenmeyer.Erlenmeyer contains synbiotic microcapsule beads and is then incubated in a water bath incubator set to 70 o C and incubated for 60 minutes.At the same time, Bacillus subtilis isolate was also incubated, which had been inoculated in nutrient broth media for 24 hours as control/free cells.

Calculation of viability of Bacillus subtilis bacteria
The bacterial viability (Bacillus subtilis) was calculated in microcapsule beads after incubation at 70 o C by adding 10 ml of microcapsule granule-breaking liquid to each treatment [19].These synbiotic microcapsule beads are then vortexed until all the beads are destroyed.After that, the synbiotic microcapsule granule solution is diluted in stages using physiological solution (SS) and then inoculated into TSA plate media.The plate is then incubated for 24-48 hours at room temperature, and colonies of bacteria growing in the dish are manually counted.The calculation of TPC (Total Plate Count) is carried out based on the formula [20] as follows:

Research design
This study design used a complete randomized design (RAL) consisting of 4

Data analysis
The data obtained were analyzed statistically by fingerprint analysis (ANOVA) using the help of the SPSS application.If Fcalculate is greater than or equal to Ftable, then followed by the Tukey difference test at the level of 95%.

Results and discussion
Synbiotic microcapsule beads using the extrusion method with alginate coating can be seen in the following figure: The beads of synbiotic microcapsules are oval to slightly oval and transparent white in color, with an increasing level of suppleness and the concentration of alginate used.The diameter of the beads of synbiotic microcapsules ranges from 0.9 -4 mm.It is still included in the microcapsule category based on the classification of microcapsule diameter by [19][20], i.e., up to 5000 μm, although the size is larger than that produced by [13].The size of the diameter of the microencapsulated beads using the extrusion method is influenced by the nozzle diameter (jet), fluid viscosity, surface tension, droplet speed, stirring speed, different material concentrations, needle dimensions, and diameters to the distance and dripping speed [20,21].The alginate used in this study was isolated from seaweed Sargassum sp.Seaweed Sargassum sp. is known to improve the performance of tiger shrimp larvae [20,21] and resistance to the WSSV virus [22,23].
The results of observations on the probiotic population in synbiotic microcapsule beads before incubation at treatment temperature were in the range of 1.23 x 10 7 cfu / mL -1.95 x 10 7 cfu / mL.The difference in alginate concentration had no significant effect on the probiotic population in synbiotic microcapsule beads.The results were obtained after the synbiotic microcapsule beads were incubated in a water bath at 70 o C for 60 minutes, as shown in Figure 3. Based on the data in Figure 3, there is a decrease in the probiotic population after incubating at a temperature of 70 o C. The highest population decrease was found in the control treatment, namely probiotic suspension cells that were not coated/free cells, which decreased by 32.35% (from 10 8 cfu / ml to 10 5 cfu / mL), followed by treatment C decreased by 27.49%, then A fell by 25.93%.The lowest treatment B only reduced by 18.05%.
The variety analysis (ANOVA) results showed that the alginate concentration significantly affected the viability of probiotics (Bacillus sp) in synbiotic microcapsule beads after incubating at 70 o C for 60 minutes.The results of further trials showed that the decrease in probiotic viability in the treatment of alginate concentrations of 1%, 2%, and 3% was not significantly different but significantly different from the control treatment without encapsulation (P<0.05).This is because probiotic cells were not encapsulated in the control treatment, so there was direct contact with relatively hot temperature conditions of 70 o C for 60 minutes.Things are different in treatments A, B, and C, where in this treatment, probiotics have been protected by alginate so that the viability of probiotics can be suppressed decrease.The microencapsulation method can maintain bacterial cells after exposure to high temperatures compared to free cells without microencapsulation [24].But giving temperature can also grip the viability of probiotics (Bacillus sp) in synbiotic microcapsules so that they decrease.This is due to conditions not synchronized with the life tolerance of Bacillus sp strains, which are optimal at temperatures around 33-55 o C and can tolerate temperatures up to 66 o C and will form spores but will experience a decrease in population [25].
Based on the results of this study, coating probiotic Bacillus subtilis using alginate can maintain the viability of probiotics so that when microcapsule beads get high-temperature stress, probiotics can still survive.This can be a reference for the application of probiotics in various aspects, such as in the manufacture of fish feed, where it is known that in the process of molding fish feed pellets, the temperature of the feed molding machine can reach temperatures of 70 o C or more, so this is no longer a barrier factor because probiotics that have been coated with alginate can maintain their viability.

Conclusion
Based on the results of the study, it can be concluded that alginate concentrations of up to 3% can reduce the rate of decline in the population of Bacillus subtilis after exposure to a temperature of 70 o C for 60 minutes so that it can be used as a reference as an effort to present probiotics that are safe and easy to apply.These data also provide an opportunity to test even higher temperature stress to obtain maximum temperature stress values against Bacillus subtilis in alginate microcapsule beads.

Fig 1 .
Fig 1.The layout of the treatment container after randomization inside the water bath incubator container.

Fig. 3 .
Fig. 3. Probiotic population in synbiotic microcapsule beads before and after incubation in an incubator water bath at 70 o C for 60 minutes.