INTRODUCTION:

Based on data from the World Health Organization (WHO) in 2008, cancer is the second largest cause of death globally, and two-third of that number comes from developing countries like Indonesia. Since there are adverse effects of treatment or anticancer drug consumption, alternative treatments or prevention of cancer is required, one of which is by using probiotics. Probiotics are living organisms which can provide health benefits when consumed in sufficient quantities [1]. The mechanism of probiotics as an anti-cancer is to detoxify genotoxins in the colon.

The colon cancer is caused by the complexity of cancer initiation, progression of cancer, and cancer exposure on the colon [2].

Lactobacillus acidophilus (1 x 10 ⁷ colony forming units / g) is LAB that has good anticancer activity because the probiotics have some anticancer mechanism by lowering DNA damage in colon cells, decreasing the activity of the procarsinogen enzyme, and improving the stimulation of the immune system [2].

In this study, Lactobacillus acidophilus probiotics are used. Besides having some anticancer mechanism, the probiotic Lactobacillus acidophilus is the most resistant to acid at around pH 2. Therefore, it is expected that probiotic remains to have high viability as it passes through the digestive tract, especially in the stomach that has an acidic pH (pH 2), since the target of probiotics consumption is the colon [3]. Alginate was chosen because this substance is a widely used biomaterial in the manufacture of a drug due to its profitability, including thickening, gelling, stabilizing and biocompatibility properties. More than 200 different alginate types have been produced to date. The ability of alginate in acid gel formation and ionotropic gel depends on pH. Alginate also provides unique properties of polymers compared to neutral macromolecules [4].

Tomato (Lycopersicum esculentum) is prebiotics to probiotics because tomatoes contain glucose, protein, fat, phosphorus, iron and vitamins that can be used as probiotic bacterial growth [5]. Tomato contains several antioxidants, one of which antioxidant marker is lycopene. The content of most lycopene in processed tomatoes is obtained in tomato paste, i.e. 42.2 mg/100g. Therefore, tomato paste has the potency to use as raw materials for the formulation [6].

Probiotics have flaws. They are unstable at acidic pH, and have high temperature and oxygen content. To improve the stability of probiotics, microparticles were prepared by microencapsulation process [7].

In the drug industry, the microparticles are used to obtain sustained-release preparations, to cover less favored odor, to improve the flow of powders, to protect the drug substance from the effects of adverse environments (moisture, oxygen, and ultraviolet light), to prevent evaporation, and to better safely handle hazardous materials. Probiotic microencapsulation is a beneficial method to improve the stability of probiotic functional food products [8]. Microencapsulation is needed not only to help probiotics survive in food products or pharmaceutical preparations, but also to protect the probiotics from gastric acid effect, in which the gastric pH can be below 2 [3].

Microencapsulation can be done by several methods such as coacervation, hot-melt, solvent evaporation cross-linking, the interfacial polymerization, spray coating, spray drying, supercritical fluid, and two other methods added by Mortazavian namely extrusion and emulsion techniques [9]. Extrusion theory is a technique in Physics for encapsulating live probiotic cells using hydrocolloids, such as alginate and carrageenan [10]. Microencapsulation with extrusion method has several advantages: it is simple and inexpensive, does not damage the probiotic cells, results in a high viability probiotics, and can be done in both aerobic and anaerobic circumstances [11].

MATERIALS AND RESEARCH METHODS:

Formula Design:

The design of microparticles made from probiotic formula and tomato paste can be seen in Table 1.

Table 1. Formula draft of Microparticles made from Probiotics and Tomato Pasta

Ingredients	Function	FI	F II	F III
Probiotics: Lactobacillus acidophilus	Active Ingredients	5 ml	5 ml	5 ml
Tomato paste	Prebiotics	1 gram	1 gram	1 gram
Natrium Alginate	Matrix	1.875 grams (2.5%)	2.25 grams (3%)	2.625 grams (3.5%)
Aquadest	Alginate solvent	70 ml	70 ml	70 ml
CaCl ₂ 1.5M	Cross linker	150 ml	150 ml	150 ml

Preparation of microparticles:

Some of the materials used in this study were obtained from Lactobacillus acidophilus PAU Gajah Mada University, and tomatoes (Lycopersicum esculentum) were obtained from plantation in Malang. Other ingredients include Natrium alginate (Pharmaceutical Grade), sterile distilled water (pro analysis), media de Man Ragosa Shorpe (MRS) broth sterile, sterile Natrium citrate (pro analysis), and the last Artemia salina obtained from Veterinary Health Republic of China.

Microparticle making was done aseptically. Probiotics were prepared as much as 5 ml from a starter with a measuring cup. Tomato paste was measured for as much as 1 gram, added to 5 ml of the probiotic starter measured earlier, and then stirred until homogeneous. Natrium alginate gel was prepared with various concentrations by dispersing Natrium alginate in 50 ml distilled water and stirring to form homogeneous gel. Natrium alginate gels were sterilized at 121 ^°C for 20 minutes before being added to a mixture of probiotic and tomato paste and stirred until homogeneous. The next stage was to take 10 ml of a mixture of probiotics, tomato paste and Natrium alginate to check the viability. Calcium chloride solution 1.5M of about 150 ml was prepared by weighing 33.075 grams of calcium chloride and dissolving it into distilled water until the volume was 150 ml. Production of microparticles was conducted by extrusion method by passing a mixture of tomato paste, probiotics, and natrium alginate onto a noozle above a container comprising calcium chloride 1.5 M solution, with a composition of 75 ml of suspension (a mixture of tomato paste, probiotic, and Natrium alginate mixture) 150 ml of calcium chloride. The noozle diameter size is 45 µm; the distance between noozle and the calcium chloride solution is 8 cm; and the compressor pressure used is 40 Psi. Perfect gelation obtained by stirring the droplets that are already in a calcium chloride solution for 2 hours at a speed of 1000 rpm. Microparticles were separated from the CaCl₂ by means of centrifugation 2500 rpm for 6 minutes, and then washed with sterile water for 3 times. The microencapsulation process was carried out at room temperature. Then the microparticles were dried by freeze dry at -80 ^°C for 20 hours.

The loss of viability after a freeze dry due to the freezing process causes the cell to lose its stability, so that it becomes more easily damaged during the drying [12]. The freeze dry process includes freezing, primary drying and secondary drying. Future primary and secondary drying process is a process of sublimation, which is the change from the solid phase directly to the gas phase that occurs at the triple point temperature of 0 to 01 ^° C and a pressure of 0.00603 atm [13]. Factors influencing the freeze dry condition are temperature and pressure [14]. Both of these affect the viability or resilience of probiotics in the microparticles, so that optimization of freeze dry conditions to obtain a high viability is required [15].

Physical Characteristics Test:

a)Morphological examination:

To see the form of microparticles, which are a combination of probiotics and tomato paste, can be observed using an inverted microscope or using an optical microscope. Samples were placed on a glass object, added with a little water, flattened and covered with a cover glass. It is important to ensure no bubbles appear after closing cover glass before observation under a microscope.

b)Determination of Particle Size:

Particle size measurements were performed by microscopy method using an optical microscope equipped with an ocular and objective micrometer. The ocular scale was calibrated by installing an ocular micrometer on a microscope and doing an observation until both scales are clearly visible. Then the starting line of the ocular scale and the starting line of the objective scale are aligned so that a precise line which coincides on both scales can be determined. After that, the ocular price was determined, and microparticles were gauged by putting them on a glass object and their diameter was measured (300 particles). The next step is grouping from the smallest to the largest size and dividing them into several intervals and classes. The distribution curve of particle size was then created [16].

c)Moisture Content Measurement:

The moisture content of microparticles was measured using Ohaus MB45 Moisture Analyzer. ON button was pressed and the device is ready. The lid was opened and the pan was cleaned before it was put in place. TARE button was pressed and the device showed zero. The sample was sprinkled into the pan (previously weighed 1-1.5 g), and the cover was closed. START button was pressed. The process took about 10 minutes before it stopped. The percentage of moisture content indicated on the instrument was then noted (Ohaus, 2001). The criterion of % MC is 1-4% [17].

Microparticles’ Viability Test:

The probiotic viability test was performed using an ALT test and performed aseptically. The ALT test procedure was as follows: microparticles were weighed as much as 1 gram and dissolved in 99 ml of 1% Natrium citrate sterile (b / v) solution at pH 6.0 and vortexed at room temperature for 1 h. It was then diluted 10 times on MRS agar and incubated at 37 ^° C in an aerobic atmosphere for 48 hours. The calculation of the number of probiotic bacteria was shown in standard colony forming units (cfu)/mL for liquids and colony forming units (cfu)/g for solids (Lin et al., 2006).

Test for Anticancer Activity:

Ten shrimp larvae were transferred into each test tube containing both the sample and the control of the solvent. Seawater was then until the volume reached 5 ml, rocked slowly to reach homogeneity, and left for 24 hours. The number of dead shrimp larvae within 24 hours was then calculated. If there is death in the control, it is corrected by Abbott's formula:

RESULTS:

This study was conducted to investigate the effect of the concentration of Natrium alginate as a matrix to physical characteristics, the viability of probiotics, and probiotic-anticancer activity of microparticles made from tomato paste using extrusion method with the concentration of Natrium alginate matrix, i.e. Formula I 2.5%, Formula II 3% and Formula III 3.5% (shown in figure 1).

The results of microparticles combination of probiotics and tomato paste of the three formulas were tested for their physical characteristics, viability of probiotics and anticancer activity. Physical characteristic tests performed include morphological form, moisture size and moisture content (MC) of microparticles. The microparticle morphology test of the three formulas was performed using inverted microscope and optical microscope with 400x magnification. The result obtained was not too spherical.

Formula I Formula II

Formula III

Figure 1. The morphology of the microparticles made from probiotic and tomato-paste formula I, II, and III using 400x magnification optical microscope

The result of the size distribution of microparticles made with inverted microscope magnification of 400x is shown in Table 2.

Table 2. The average particle size of probiotic-tomato paste

Group	Average Size (μm)
Formula I	34.60
Formula II	34.69
Formula III	80.53

According to Brun-Graeppi, factors that affect the size of the microparticles are polymer solution concentration or matrix, viscosity, and the distance between the hatching to the surface of the cross-linking solution. Low concentration of Natrium alginate will result in smaller size or diameter of microparticles.

The next test is characteristics of moisture content (MC), and the results obtained is shown in Table 3.

Table 3. Moisture Content (MC) of microparticles

Group	MC (%)
Formula I	16.05 ± 0.41
Formula II	13.34 ± 0.23
Formula III	12.65 ± 0.32

After the MC was tested with One-Way ANOVA, significant differences (p <0.05) were found between formula I, formula II, and formula III, but there were no significant differences between formula II and formula III (p> 0.05).

To determine the influence of Natrium alginate concentration on the viability of the microparticles, it is necessary to carry out viability test before extrusion by calculating Total Plate Count (ALT) in each formula. This is to ensure that the probiotics used are already qualified, i.e. > 10 ⁷ cfu/g. In addition, ALT calculation before extrusion also aimed to determine the efficiency of trapping the microparticles made from probiotics and tomato paste with varying concentrations.

Table 4. Comparison of ALT Log Lactobacillus acidophilus

Group	Log ALT (cfu / g)
Group	before microparticle	after microparticle
Formula I	8.57 ± 0.04	7.27 ± 0.11
Formula II	8.68 ± 0.22	7.69 ± 0.01
Formula III	8.60 ± 0.08	8.22 ± 0.08

After the formula was made in form of microparticles by extrusion method and dried by freeze dry method for 20 hours at a temperature of -80°C, the percentage of viability after freeze dry was obtained compared to the viability of the original formula as shown in Table 5.

Table 5. Viability of Lactobacillus acidophilus

Group	Viability of the microparticles process (%)
Formula I	84.83
Formula II	88,59
Formula III	95,58

From the viability calculation formula of the three formulas, One-Way ANOVA statistical test was then performed. It was found that the result of formula I has a significant difference (p <0.05) from formula III, while there was no significant difference in viability (p> 0.05) between formula II to formula I and III.

BST test results of observational data were processed by probit regression analysis using SPSS 20. The analysis of the data generated the value of LC ₅₀ (concentration required for shrimp larvae mortality by 50%) which correlate to the number of dead shrimp larvae at a concentration of the test solution. After the test results of the anticancer activity of dried microparticles were analyzed using probit regression, LC ₅₀ values are obtained as shown in Table 6.

Table 6. Results of LC ₅₀ values of microparticles using SPSS probit analysis

Replication	LC ₅₀ (ppm)
Replication	Formula I	Formula II	Formula III
1	289.58	267.97	208.18
2	412.55	374.49	309.86
3	289.58	288.64	174.34
Average ± SD	330.57 ± 70.99	310.37 ± 56.49	230.79 ± 70.53

Statistical analysis, which is One-Way ANOVA method with Tukey HSD test in SPSS 20, was used to compare LC ₅₀ values. The value obtained was p> 0.05, so there is no significant difference of the three formulas. In addition, it can also be seen from the value of F count = 2.908. The value of F count (2,908) <F table (4.4590), so it is said that the anticancer activity between the three formulas before extrusion has no significant difference.

DISCUSSION:

Generally, microparticles with the matrix of Natrium alginate will result in a smooth form, but irregular or spherical form can happen if the rate or speed of spraying in the extrusion process is too slow. It is thus advisable to do the optimization rate of spraying during the extrusion process in order to obtain spherical microparticles. Spherical shape is expected in the formation of these microparticles because spherical shape allows good flow rate of microparticles. If these microparticles are produced, they will simplify the process of filling into the packaging.

In this study, the factor affecting the size of the microparticles is Natrium alginate concentration because other conditions were made equal. The greater the concentration of Natrium alginate, the more cross-linked reactions occur, causing bigger size of the microparticles. In addition, the greater the concentration of the matrix, the greater the viscosity generated, increasing the particle size. Of the three formulas in this study, particles obtained are in the size range of microparticles that is 1-1000 μm.

Increasing the matrix concentration may increase the MC value, but this study shows that the higher the Natrium alginate concentration, the smaller the MC value. When the Natrium alginate is higher, the size is getting bigger so the total surface area is getting smaller and the ability to absorb moisture is less because one of the properties of calcium alginate is hygroscopic. The optimum value of MC for microparticles of probiotics is 1-4%, while the three formulas obtained MC> 4%. Optimization is required to dry the microparticles. One of the methods that can be done is vacuum drying or oven at a temperature of 40 ⁰C by optimizing the drying time. 40 ⁰C is the optimum temperature for the bacteria Lactobacillus acidophilus. The bacteria are also sensitive to high temperatures or above 50 ⁰C so it is advisable to set the oven temperature to 40 ⁰C to maintain the viability of Lactobacillus acidophilus. Another advantage of vacuum drying method at 40 ⁰C is obtaining MC value by 1-4%. Besides, the cost required is also reduced to one-third of the costs incurred when using freeze dry method. After drying by means of oven temperature of 40⁰C for 16 hours, the MC value of 6.32% was obtained. This could be considered for future research: MC 1-4% can be done by means of oven with a temperature of 40 ⁰C for> 16 hours.

Increased natrium alginate concentration will increase the efficiency of probiotic viability trapping. The greater the concentration of natrium alginate, the greater the cross-linking process, and the ability to trap probiotics in the microparticles becomes stronger so that the viability of probiotics after extrusion is larger. The percentage of viability increases with the amount of Natrium alginate concentration used in microparticles and ALT values. Probiotics in the microparticles of the three formulas have a value of> 10 ⁷ cfu / g, which is a requirement for the amount of probiotics used as anticancer.

Increased concentrations of alginate matrix in the microencapsulation process could improve the viability of probiotic Lactobacillus acidophilus to 10 ¹ -10 ⁷ cfu / g. It can be concluded that a 0.5% increase in Natrium alginate concentration does not increase the viability of probiotics, but with an increase in natrium alginate concentration of 1% from 2.5% to 3.5% may increase the viability of the probiotics-tomato paste microparticles.

The test to find the effect of Natrium alginate of microparticles made from probiotics and tomato paste on the anticancer activity was conducted by using Brine Shrimp Lethality Test (BST) or toxicity testing on shrimp larvae. Shrimp larvae used in this study is Artemia Salina. Artemia Salina was used because the cell walls of Artemia Salina resemble cancer cells, whereas seawater habitat or media used is sea water with temperature of about 27-29 ⁰ C and pH between 7.00 - 7.50. Temperature and pH must be considered because the condition is the optimal condition for the process of hatching shrimp eggs into shrimp larvae.

BST test is done by making various concentrations of each formula. The concentrations used were 10, 20, 100, 200, 500, and 1000 ppm. A material is said to have anticancer activity if the concentration is <1000 ppm; thus the various concentrations were made. The solvent used to dissolve the microparticles is natrium citrate because these substances can dissolve the calcium alginate, which is the result of a cross-linking between natrium alginate and calcium alginate. The BST test used 10 Artemia Salina shrimp larvae at each observation, in which the focus is observing the number of dead shrimp larvae. The more death of shrimp larvae observed, the higher the anticancer activity. To determine the influence of solvent activity and matrix from microparticles, a negative control of microparticles mas made without probiotics and tomato paste. From the research, it is found that there was one dead shrimp larvae which should be corrected using Abbot's formula. These results differ from the results expected in viability result which has significant increase in formula I on formula III. This means that an increase in viability of 10.75% is not sufficient to increase anticancer activity.

From the test results of physical characteristics, viability, and anticancer activity, it can be concluded that with the presence of increasing concentrations of natrium alginate (2.5%, 3% and 3.5%), the anticancer activity of microparticles made from probiotics and the tomato paste is the same, although the physical characteristics test and viability test showed an increase from formula I to formula III. Based on these results, the production process should use the smallest concentrations of natrium alginate, which is formula I (concentration of natrium alginate 2.5%) because it is more cost effective. Microparticles made from probiotics and tomato paste with a concentration of 2.5% natrium alginate have anticancer activity equal to the concentration of natrium alginate 3% and 3.5%.

CONCLUSION:

From this research, several conclusions can be drawn. First, an increase in the concentration of natrium alginate (2.5%, 3% and 3.5%) resulted in an increase in the size of the microparticles made from a combination of probiotics and tomato paste. Second, an increase in concentrations of natrium alginate from 2.5% to 3% Decreased the moisture content of the microparticles made from combination of probiotics and tomato paste, while from 3% to 3.5% has the same moisture content. Thirdly, increased concentration of natrium alginate for 1%, which is from 2.5% to 3.5%, increases the viability of microparticles made frm combination of probiotics and tomato paste. Finally, increased natrium alginate concentration of 2.5%, 3%, and 3.5% in the manufacture of microparticles made from a combination of probiotics and tomato paste does not affect the anticancer activity.

CONFLICT OF INTEREST:

There is no conflict of interest

REFERENCES:

1 FAO/WHO. Guidelines for the Evaluation of Probiotics in Food. Food and Agriculture Organization of the United Nations/World Health Organization, Canada. 2002.

2 Wollowski I, Rechkemmer G and Zobel BLP. Protective role of probiotics and prebiotics in colon cancer. Am J Clin Nutr. 2001; 73 (suppl); 4–5.

3 Ding WK, Shah NP. Effect Of Various Encapsulatingmaterials On The Stability Of Probiotic Bacteria. Journal Of Food Science. 2009; 74; M100-M107.

4 Kamrun Nahar, Md Kamal Hossain, Tanveer Ahmed Khan. Alginates: The Wonder Molecule and its Gelling Techniques. Research Journal of Pharmacy and Technology. 2017; 10(9): 3195-3204.

5 Chottanom, Pheeraya. Pulsed Vacuum Osmotic Dehydration of Cherry Tomatoes: Impact on Physicochemical Properties and Probiotics Entrapment. Available from: http://wjst.wu.ac.th/index.php/wjst/article/view/1765

6 Tsang G. Lycopene in Tomatoes and Prostate Cancer. Available from: http://www.healthcastle.com

7 Gbassi GK and Vandamme T. Probiotic Encapsulation Technology: From Microencapsulation to Release into the Gut. Available from: http://doi.org/10.3390/pharmaceutics4010149.

8 Kailasapathy K. Microencapsulation of probiotic bacteria: technology and potential applications. Current Issues in intestinal Microbiology. 2002; 3(2): 39–48.

9 Kinam Park and Yoon Yeo. Encyclopedia Of Pharmaceutical Technology Third Edition. 2007: 2315-2327

10 Mortazavian A, Razavi SH, Ehasani MR, Sohrabvandi S. Principle and methods of microencapsulation of probiotic microorganisms. Iranian Journal of Biotechnology. 2007; 5(1): 1-18.

11 Burgain C, Gaiani M, Linder J, Scher, Encapsulation of probiotic living cells: From laboratory scale to industrial Applications. J. Food Eng. 2011;104: 467–483.

12 Solanki HK, Pawar DD, Dushyant AS, Prajapati VD, Jani GK, Mulla AM, Thakar PM. Development of Microencapsulation Delivery System for Long-Term Preservation of Probiotis as Biotherapeutics Agent. BioMed Research International. 2013; 1-21.

13 Johnson JAC and Etzel MR. Properties of Lactobacillus helveticus CNRZ-32 attenuated by spray drying, freeze drying, or freezing. J. Dairy Sci. 1995; 78: 761-768.

14 Nireesha GR, Divya L, Sowrnya C, Venkateshan N, Niranjan Babu M, Lavakumar V. Lyophilization/Freeze Drying – An Review. International Journal of Novel Trends in Pharmaceutical Sciences. 2013; 3(4).

15 Chaline Caren Coghetto. Probiotics production and alternative encapsulation methodologies to improve their viabilities under adverse environmental conditions. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27456038

16 Martin M. Surfactans and Polymers in Drug Delivery. Marcel Dekker Inc , New York. 2002.

17 Parrot EL. Pharmaceutical Technology: Fundamental Pharmaceutic. Burgess, London. 1970.

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Received on 12.02.2018 Modified on 02.03.2018

Research J. Pharm. and Tech 2018; 11(6): 2575-2580.

DOI: 10.5958/0974-360X.2018.00476.6