Abstract: Objective: The aim of this study was to test the ability of Lactobacillus brevis and Lactobacillus plantarum derived from fermented cabbage waste juice to acts as probiotics. Materials and Methods: Tests of probiotic ability included tests of bile salt resistance and pH resistance and tests of sensitive inhibition of Escherichia coli and Salmonella pullorum growth. Results: Lactobacillus brevis and Lactobacillus plantarum derived from fermented cabbage waste juice were able to grow and develop at pH values from 2.5-5.5 and bile salt concentrations of 1-5%. Lactobacillus brevis was able to strongly inhibit Escherichia coli and Salmonella pullorum growth, while Lactobacillus plantarum showed very potent inhibition of Escherichia coli growth and potent inhibition of Salmonella pullorum growth. Conclusion: Lactobacillus brevis and Lactobacillus plantarum derived from fermented cabbage waste juice are suitable for use as poultry probiotics.
INTRODUCTION
Cabbage is a type of vegetable that grows in the highlands of Indonesia. Cabbages wilt, get damaged and decay easily, leading to a foul odor that causes environmental problems. Cabbage waste is commonly found in traditional, untapped markets. The use of cabbage waste as probiotics had not been previously studied, which inspired researchers to test Lactobacillus brevis (L. brevis) and Lactobacillus plantarum (L. plantarum) as poultry probiotics. L. brevis and L. plantarum which were tested as probiotics are derived from the juice of fermented cabbage waste. Utama et al.1 and Plengvidhya et al.2 stated that cabbage processed by fermentation contains microbes such as Leuconostoc mesenteroides, Pediococcus pentosaceus, Rhizopus oryzae and Saccharomyces cerevisiae. Cabbage waste is a waste product from vegetable markets and consists of outer shells of cabbages damaged by impact and collision, which make these cabbages not worth selling. The amount of waste generated is as much as 3-5% of the total weight of cabbage3. Cabbage waste can cause pollution, so there is a need to develop methods to handle and process this waste in order to use it as a probiotic.
Probiotics are live microbes that are administered into the gastrointestinal tract and provide benefits to the digestive tract of the host. Fuller4 and Mateova et al.5 explained that probiotics supplementary foods that are composed of microbes that are beneficial to and affect the host by improving the microbial balance in the digestive tract. Probiotic bacteria usually belong to the genera Lactobacillus and Bifidobacterium. Probiotics must exhibit the following characteristics: Ability to live at low pH, resistance to bile salts, production of toxins, live cell density of more than 106 CFU mL–1, ability to survive and perform metabolic activities in the digestive tract and ability to survive during storage, in addition probiotics should not trigger an immune response6,7.
Some types of probiotics can produce natural antibiotics that can inhibit the growth of pathogenic microbes. Probiotics can help stabilize intestinal microbes, improve the permeability of the intestinal barrier and enhance systems and responses of mucosal IgA that prepare the intestinal mucosal barrier against harmful microbial infections and infectious agents. Wolfenden et al.8 stated that administering probiotics in the form of an effective competitive exclusion (CE) culture can reduce colonization by the pathogenic microbe Salmonella enteriditis (SE) in the digestive tracts of broiler chickens. The use of probiotics in chickens is reported to decrease urease activity and as a result, the formation of ammonia is reduced9. The use of probiotics in chickens increases the rate of growth and use of nitrogen, enhances immunity to infection and increases egg production10,11. The results of this study are consistent with the research by Mateova et al.5, who stated that the use of probiotics increases the weight of broiler chickens. Several researchers have reported positive in vivo effects such as strengthened mucus production, activated macrophages in the presence of Lactobacillus, secretory IgA stimulation, proinflammatory enhancement and cytokine production12. Thus, further studies are needed to create safe probiotics for poultry and to enhance the production of antibiotic-free livestock. To this end the ability of the probiotics L. brevis and L. plantarum derived from fermented cabbage waste juice was tested to act as poultry probiotics.
MATERIALS AND METHODS
The materials used in this study were L. brevis and L. plantarum isolates from fermented cabbage waste, de Man, Rogosa and Sharpe (MRS) medium (Oxoid, UK#CM 0361), de Man, Rogosa and Sharpe (MRS) medium (Oxoid, UK #CM 0359), Aquades, physiological saline, HCl, bile salts, Mueller-Hinton agar (MHA) medium (Oxoid,UK #CM 0337) and Escherichia coli and Salmonella pullorum isolates. The tools and instruments used in this study were the following: Electric benchtop autoclave sterilizer (All American, USA), incubator (Memmert, Germany), digital scales (Ohaus, USA), glass Erlenmeyer flasks (Schott Duran, Germany), measuring cup (Schott Duran, Germany) and pH meter (Crison, Spain).
Lactobacillus brevis and Lactobacillus plantarum were obtained from the fermented cabbage waste juice. Fermented cabbage waste juice was produced by cutting of the cabbage waste as smoothly as possible. The cabbage waste was then blended and added with 8% of salt (without iodine) and 6.7% of molasses. Subsequently, the mixture was spontaneously fermented in a closed container for 6 days. The fermented products were then used as a source of the above mentioned bacteria. The study started by reviving the Lactobacillus brevis and Lactobacillus plantarum isolates, which were ordered as agar slants. The ability of the lactic acid bacteria to acts as probiotics was tested by pH and bile salt resistance tests and by testing the sensitivity of inhibition of Escherichia coli and Salmonella pullorum growth. The tests were conducted as follows:
| • | pH resistance test: The pH resistance was tested done by using the method described by Taheri et al.13 with some modifications. The aim of this test was to determine the resistance of L. brevis and L. plantarum isolates grown in acidic media or at various pH levels. A total of 1 mL of L. brevis or L. plantarum cultures was grown in 10 mL of MRS broth medium at pH 2.5, 3.5, 4.5 and 5.5 at 37°C for 12 h. The growth of the microorganisms was detected as the presence of turbidity in the medium and the absorbance of the cultures at a wavelength of 600 nm was quantitatively measured by using a spectrophotometer. Optical density (OD) was determined by the following formula: |
OD = (OD600 grown cultures-OD600 blank)
| • | Bile salt resistance test: Bile salt resistance was tested by using the method described by Taheri et al.13 with some modifications. This aim of this test was to determine the resistance of L. brevis and L. plantarum isolates to the level of bile salts in the growth medium. The resistance of L. brevis and L. plantarum in bile-salt-containing medium was tested by adding bile salts at concentrations of 1, 2, 3, 4 and 5% into tubes containing MRS broth. A total of 1 mL of L. brevis or L. plantarum cultures was added to 10 mL of medium and the cultures were incubated at 37°C for 12 h. The growth of the microorganisms was detected as the presence of turbidity in the medium and the absorbance of the cultures at a wavelength of 600 nm was quantitatively measured by using a spectrophotometer. Optical density (OD) was determined by using the following formula: |
OD = (OD600 grown cultures-OD600 blank)
Sensitivity test of Escherichia coli inhibition: This test was conducted to determine the ability of L. brevis and L. plantarum isolates to inhibiting the growth of Escherichia coli. The method used was a modified dual-culture method. Escherichia coli was grown in petri dishes on MHA and the agar was then perforated using a thin hole tool to make a 1 cm diameter incision. L. brevis or L. plantarum isolates were inoculated into the hole at a volume of 250 μL and the plates were incubated for 24 h at 37°C. After incubation, the observed barrier zone was measured using a ruler14.
Sensitivity test of Salmonella pullorum inhibition: This test was performed to determine the ability of L. brevis and L. plantarum isolates to inhibit the growth of Salmonella pullorum. The method used was a modified dual-culture method. Salmonella pullorum was grown in petri dishes on MHA and the agar was then perforated using a thin hole tool to make a 1 cm diameter incision. L. brevis and L. plantarum isolates were inoculated into the hole at a volume of 250 μL and the plates were incubated for 24 h at 37°C. After incubation, the observed barrier zone was measured using a ruler14.
Non-parametric statistical analysis: The data from the pH resistance test, bile-salt resistance test and sensitivity tests of Escherichia coli and Salmonella pullorum inhibition were analyzed by non-parametric statistical analysis15.
RESULTS AND DISCUSSION
Resistance of Lactobacillus brevis and Lactobacillus plantarum to pH: The resistance of L. brevis and L. plantarum to pH is shown by the data in Table 1. Table 1 shows the ability of L. brevis and L. plantarum to survive in pH values from 2.5-5.5. Overall, the isolation and identification of lactic acid bacteria from the fermented cabbage waste juice yielded 8 isolates but upon reinoculation, only 2 isolates exhibited consistent growth. This observation was a result of the addition of salt as much as 8% of the fresh weight of cabbage juice, during the fermentation process. The addition of salt to the media was intended to serve as selection method and inhibit the growth of pathogenic bacteria during fermentation. The amount of lactic acid bacteria in the fermented cabbage waste juice was 2×1010 CFU mL–1 and the total fungal growth was 29×108 CFU mL–1. The secondary metabolite content and pH were as follows: pH, 3.46 total acid 1.10%, acetic acid 0.02%, butyric acid 0.002% and lactic acid 0.80%.
Lactobacillus brevis and L. plantarum isolates have the ability to survive in low-pH environments. The L. brevis and L. plantarum isolates showed resistance to a pH of 2.5 for 12 h (Table 1). The populations of the L. brevis and L. plantarum isolates were greater than 108 CFU mL–1 (Table 1), which proved that the ability of L. brevis and L. plantarum isolates to grow at low pH values is very good, indicating that these bacteria could survive in the digestive tracts of poultry. Saarela et al.16 stated that resistance to low pH is an important characteristic of probiotics. The recommended concentration of lactic acid bacteria as poultry probiotics is 108 CFU kg–1 of feed in order for the bacteria to survive in the digestive tract17. Table 1 shows that the higher the pH concentration is the more the microbes grow. Purwoko18 stated that bacterial growth can be determined by measuring the difference in absorbance before and after incubation.
| Table 1: | Optical density (OD) of Lactobacillus brevis and Lactobacillus plantarum isolates at various pH values* |
| *Growth in MRS broth medium with an incubation period of 12 h, SEM: Standard error of the treatment means | |
| Table 2: | Optical density (OD) of Lactobacillus brevis and Lactobacillus plantarum isolates in various bile salt concentrations* |
| *Growth in MRS broth medium with an incubation period of 12 h, SEM: Standard error of the treatment means | |
The number of bacterial cells can be measured by determining the turbidity of the culture, the more turbid a culture is, the greater the number of cells.
Resistance of Lactobacillus brevis and Lactobacillus plantarum to bile-salt concentration: The resistance of L. brevis and L. plantarum to various concentrations of bile salts is shown by the data in Table 2. The results showed that all isolates were able to grow in media containing bile salts at concentrations from 1-5% for 12 h.
Bile salts are amphipathic compounds, one end is soluble in water (polar/hydrophilic) and the other end is not water soluble (nonpolar/hydrophobic). This amphipathic structure causes the bile salts to emulsify fat and directly affect the life of microorganisms in the digestive tract, especially in the small intestine. The L. brevis and L. plantarum isolates had a life span of 12 h at a bile salt concentration of 5% (Table 2). The higher the concentration of bile salts is, the greater the population sizes of L. brevis and L. plantarum. Prasad et al.19 stated that bile salts are secreted by the small intestine and are environmental stress factors for microbial growth. Bile salts inhibit microbial growth by weakening microbial cell membranes, the main components of which are fats and fatty acids that can be damaged by bile salts.
Bile salts in the small intestine can also be said to affect microorganisms as "biological detergents", i.e., fluids that have the ability to dissolve phospholipids, cholesterol and proteins. Most of these compounds can rearrange cell membranes, thus causing microbial cell lysis. At high concentrations, bile salts are very toxic antimicrobial substances20. Table 2 shows that the L. brevis and L. plantarum isolates were able to grow well in bile salt concentrations of up to 5%, with the population size staying above the McFarland standard of 108 CFU mL–1.
| Table 3: | Results of sensitivity test of Escherichia coli and Salmonella pullorum growth inhibition* |
*The method used was a modified dual-culture method in Mueller-Hinton agar medium with an incubation period of 24 h | |
Bezkorovainy21 stated that bile in the small intestine inhibits the growth of existing microbes, therefore, for L. brevis and L. plantarum isolates to be probiotics, these bacteria should be able to withstand bile salts in order to survive and perform probiotic function in poultry intestines. Resistance to bile salts is an important characteristic of probiotics.
Sensitivity test of Escherichia coli and Salmonella pullorum growth inhibition: Davis and Stout22 stated that in terms of antibacterial potency, clear zones of 20 mm indicates very strong inhibition, 10-20 mm indicates strong inhibition, 5-10 mm indicates moderate inhibition and 5 mm or less indicates weak inhibition. The L. brevis isolate showed strong inhibition of Escherichia coli and Salmonella pullorum growth, while L. plantarum showed very strong inhibition of Escherichia coli growth and strong inhibition of Salmonella pullorum growth (Table 3). L. brevis produces a natural antibiotic called lactobrevin and L. plantarum produces lactolin, which can inhibit the growth of pathogenic bacteria.
Lactobacillus sp. ferment carbohydrates to produce lactic acid, which can lower the pH. Acidic pH can inhibit the growth of microbes, especially microbial pathogens23. Murry et al.24 showed that pure cultures of L. plantarum produce lactic acid at high quantities in vitro, which can decrease the pH, thus inhibiting the growth of pathogenic bacteria. Lactobacillus sp. can inhibit the growth of pathogenic bacteria by blocking receptors from pathogenic bacteria and preventing these bacteria from colonizing the intestines or by removing toxic metabolites produced by the pathogenic microbes25. Lactic acid bacteria produce a wide variety of antimicrobial components: hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl (butane-2,3-Dione) and bacteriocin. All of these components are antagonistic to the growth of pathogenic bacteria26. Therefore, the use of L. brevis and L. plantarum isolates derived from fermented cabbage waste juice as poultry probiotics is very feasible as these bacteria exhibited endurance at low pH and in bile salts and exhibited sensitive inhibition of Escherichia coli and Salmonella pullorum.
In this study, a new type of probiotic was found, derived from fermented cabbage waste juice that can be beneficial to poultry. This study can be used as a scientific reference by researchers as there has been no previous research on probiotics derived from cabbage waste. For the poultry industry, this study describes the innovation of technology to derive probiotics from sources are readily available, making the process cheap and environmentally friendly. Thus, this invention can be exploited by all parties to create poultry farms that are free of antibiotics.
CONCLUSION
Lactobacillus brevis and Lactobacillus plantarum are suitable for use as poultry probiotics, which is evident from the ability of these bacteria to grow and develop at low pH (2.5-5.5) and in bile salts at concentration of 1-5%. L. brevis demonstrated strong inhibition of Escherichia coli and Salmonella pullorum growth, while L. plantarum showed very strong inhibition of Escherichia coli growth and strong inhibition of Salmonella pullorum growth.
ACKNOWLEDGMENT
The authors of this study are grateful to the Directorate of Research and Community Service (DRPM), Ministry Research, Technology and High Education, Indonesia for the facilitation of this research by funding of doctoral dissertation research in 2018.