Dengke naniura source of probiotics to lower cholesterol

Dengke naniura is one of the traditional foods found in Batak Toba, made from carp processed without cooking, but only by soaking it in an acid solution for 7 hours and adding spices. Objective of this research is to acquire lactic acid bacteria from dengke naniura as a candidate probiotic that can lower cholesterol. Out of 20 isolates isolated from dengke naniura and characterized as lactic acid bacteria. In accordance with the test results, 15 isolates were non-motile, rod-shaped, Gram-positive catalase negative, and capable of growing at pH 3.0-8.5. Seventeen isolates were tested for their efficacy their ability to avert the growth of pathogenic bacteria and lowering cholesterol in the media. Inhibition of lactic acid bacteria isolates against Salmonella typhimurium IFO 12529 with a range of 5.9-9 mm, Escherichia coli IFO 3301 by 5.9-10.7 mm, and Staphylococcus aureus IFO 13276 with a range of 3.9-7.9 mm. All isolates tested were able to reduce cholesterol in the media by 34-65%. The results of this study are expected to have an impact on the provision of agricultural commodities in the form of processed products that are more diverse and have a health impact, especially for farmers.


Introduction
Probiotics are live microbia that give the inang health benefits when given in sufficient doses [1].To screen microorganisms as probiotic candidates, they should to be secure and capable of surviving in bile salts and stomach fluid, attach to the epithelial cells and mucosal surface, and have antibacterial properties to against potentially bacteria pathogens.The most commonly used probiotic microorganism is lactic acid bacteria (LAB), because of its high survival rate in the human digestive system and its safe use in food.
LAB are usually thought of as beneficial microorganisms and have long been related to food and feed fermentations; some strains even function as probiotics, or bacteria that promote health [2].Lactic acid bacteria have been involved in certain fermented foods, including fruit, vegetables, milk, and meat.Usually food during the fermentation process produces several compounds such as acid-producing bacteria, which convert the sugars into organic acids.However, in addition to producing organic acids that are antagonistic towards phatogen bacteria, also produce bacteriocin, hidrogen peroxide (H2O2), diacetyl (C4H6O2), and other secondary metabolites [3].Lactic acid bacteria are found in various foods originating from Indonesia, as in fruit fermentation, vegetable fermentation, milk fermentation, cassava tree and glutinous rice fermentation, tempeh fermentation, moromi, and fish fermentation [4,5,6,7,8].Prior research revealed that the LAB found in various spontaneous fermented foods in Indonesia exhibited their potential as probiotics such as Lactobacillus plantarum from gatot dadih [9].
Dengke naniura is one of the Batak Toba traditional foods in North Sumatera, which is prepared from carp (Cyprinus carpio) without cooking.The processing of dengke naniura is done by soaking fish meat in jungga lime juice and adding spices to obtain tender fish meat.Soaking in jugga lime juice will turn raw fish into a fishy non-tasting, and the use of various spices makes dengke naniura more delicious [10,11].Previous studies showed that the naniura contains lactic acid bacteria [12,13].Study isolation LAB from naniura and potential to reduce cholesterol has not been conducted.
Cholesterol is the fat fraction in mammals.Generally, this component is formed in the liver when there are foods, especially those containing saturated fat [14].Cholesterol serves as a support for cells to precursors from various synthesis of compounds such as vitamin D, steroid hormones, and reproductive hormones, to important compounds in fat production such as bile salts.But in certain circumstances, a high cholesterol level in the blood can have a detrimental effect on humans, such as heart disease.Decreasing serum cholesterol may reduce the incidence of coronary thrombosis in individuals with hypercholesterolemia [15].Several studies have indicated that lactic acid bacteria have potential as probiotic agents to lower cholesterol [16,17,18,19,20,21].
Goal 2 of the sustainable development agenda, which seeks to enhance nutrition, end hunger, provide food security, and promote sustainable agriculture, is particularly relevant to the study findings discussed in this article.In the hopes that it can advance the growth of the food supply, particularly useful food with native probiotic microorganisms from dengke naniura.

Sampling and bacterial strain used
Isolated lactic acid bacteria from dengke naniura.The pathogenic strains used for this research were Salmonella tiphymurium IFO 12529, Escherichia coli IFO 3301, and Staphylococcus aureus IFO 13276, that acquired from the Culture Collection of Food and Nutrition, Center for Food and#Nutrition Studies, Universitas Gajdah Mada.

Isolation and characterization of lactic acid bacteria
The term lactic acid bacteria isolation refers to [22].Ten grams dengke naniura was added to 90 milliliters of MRS broth medium, and homogenized by crushing it with a stomatcher (Seward 400, England), and put in incubator temperature 37°C let 24 hours.The solution was diluted from 10 -2 to 10 -8 using MRS broth (from Merck, Germany).A patridish containing sterilized MRS agar (from Oxoid), calcium carbonate (CaCO3) as much as 1%, and Na azida 10 ppm was filled with up to 1 mL of dilutions by use the pour-plate technique.After that, the dish was incubated for 48 hours at 37°C.Based on size, color, and shape, colonies that were encircled by a distinct zone were separated.The isolate was cultivated for 48 hours at 37°C and purified using the spread plate technique on MRS agar.Up until a pure culture was achieved, the purification procedure was conducted.As a first step in looking for lactic acid bacteria, cell shape, Gram staining, motility, catalase test, and acid tolerance were employed [7].Gram staining was determined by the general method.Catalase reaction by pouring the young culture of lactic acid bacteria with H2O2 solution, the reaction of catalase is indicated by the production of gas CO2 (bubble).Motility was detected by growing the culture on MRS soft agar.Effects of different pH starting at 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8,5.9.0, and 9.5 were detected by growth in MRS broth.All the LAB isolates were stored at -20°C in an eppendof tube with a 1:1 ratio of 10% skim and 20% glycerol.Stock cultures were recultivated in a broth made from MRS and cultured for 24 hours at 37°C prior to testing.

Assay for antibacterial activity in lactic acid bacteria
S. tiphymurium IFO 12529, E. coli IFO 3301, and S. aureus IFO 13276 were all pathogenic strains tested in this study.They came from Universitas Gajdah Mada's Center for Food and Nutrition Studies' Food and Nutrition Culture.Well diffusion methods were used to evaluate the antibacterial activity [9].Bacteria were grown for 24 h in Muller-Hilton agar (Difco, USA).To the petri dish was added 0.1 of culture.Melting Muller-Hilton agar media (warm temperature 40°C) was added to the petri dish with the pour plate method.The petri dish was stored in a cool room (4°C) for 1-1.5 h.Then the media was plugged for making wells (three wells for each petridish) with a diameter of 6 mm.Then, 50 µL of 24 h LAB cultures were dropped in every well, and the petridishes were stored in low temperature room for 2-3 hours for the diffusion of the metabolites of the cultures, then incubated for 24 hours at 37°C.The ability to inhibit the growth of pathogens was shown by the appearance of clear zone surrounding the well.The diameter of the well is 6 mm.The antibacterial ability of LAB waere determined by the diameter of clear zones minus the diameter of wells in millimeters.

Cholesterol assay
A modification of the broth system utilized [24] was used for this study.As a bile salt, 0.4% (w/v) oxgall was added to freshly made 0.2% (w/v) sodium thioglycollate and MRS broth is called MRS-THIO broth.One oxygen scavenger that was employed was sodium thioglycollate.After filtration (0.22 μmpore-size filter) to sterilize it, alcohol-soluble cholesterol was dded at a final concentration of 100 μg/ml to the broth.For 24 h, the broths containing 1% of each strain were incubated anaerobically at 37°C in a Forma Anaerobic System (from Thermo Electron Corporation, USA).Centrifugation was used to extract the cells from the culture broth for 10 minutes at 12,000 g and 4°C.To determine the cholesterol in the media by using the following process.
A total of 3 mL of 95% ethanol and 2 mL of 50% potassium hydroxide were added to 1 mL of samples.After adding each component, the contents of the tubes were combined and heated for ten minutes in a water bath at sixty degrees Celsius.Each tube was filled with 5 mL of hexane and properly mixed after cooling.To enable phase separation, after adding and swirling a milliliter of distilled water sample, the tubes were allowed to stand at room temperature.Three milliliters of an aliquot sample of the hexane layer were put in a sterile tube, and nitrogen gas was used to evaporate the hexane.Each tube acquired a 4 mL sample of o-phthalaldehyde reagent, and the tubes were left to stand at room temperature for ten minutes.Then 2 mL of concentrated sulfuric acid was added and allowed to stand for 10 minutes, the absorbance at 550 nm was measured in comparison to a reagent blank.The values of absorbance were compared to the results using standards for cholesterol.

Statistical analysis
Every test has been carried out three times, and the values were presented as the mean.The statistical analysis sofware used the SPSS version 21.Variance analysis was used to evaluate the data prior to when necessary, the Post-Hoc LSD test at a significance threshold of p>005.

Lactic acid bacteria isolated
Dengke Naniura was used to isolate lactic acid bacteria.A clear zone surrounded the colonies revealed the presence of twenty isolates, including LAB.The appearance of the clear zone is caused by the acid agent dissolving the CaCO3 on the MRS medium [25].These isolates were all rods and cocci, Gram positive, non-motile, and could grow at pH 3.0-9.0.The catalase test showed that fifteen isolates reacted negatively to catalase and three positively to catalase.Lactic acid bacteria are characterized by their rodshaped or cocci shape, Gram positive, catalase negative, non-motile, and able to grow at low acidity.Isolates that have characteristics of LAB are used to evaluate the inhibitory effect on pathogenic bacteria and cholesterol reduction tests.The results of the study [26], revealed that 28 different lactic acid bacteria were found in a variety of Indonesian fermented foods such as growol, gatot, tape, tempoyak, pickle, and tempe were Gram positive, rods, non-motile and non-sporing, and nd a negative catalase response.After LAB isolated from bakasang, 27 isolates rods and cocci that were Gram positive.The cells did not motile, non sporing, and they did not respond well to catalase [5].

Antimicrobial activity
Table 1 contains a summary of the antibacterial activity of LAB isolates against pathogenic bacteria.The result show that all isolates LAB were able to inhibit the three pathogenic bacteria, i.e., Salmonella tiphymurium, Escherichia coli, Staphylococcus aureus.The inhibitory zones' sizes varied and ranged between 5.9-9.5 mm; 5.9-10.7 mm; dan 3.9-7.9mm for Salmonella tiphymurium, Escherichia coli, Staphylococcus aureus respectively.The highest activity against Salmonella tiphymurium shown by RS-2 and RS-15 with 9.5 mm diameter inhibitor zone, activity against Eschericia coli by RS-15 with 10.7 mm diameter inhibitor zone and activity against Staphylococcus aureus by RS-2 and RS-18 with 7.9 mm diameter inhibitor zone.6.3 a 7.9 c 4.8 c Note: * The significant difference (p<0.05) according to Duncan's multiple range test between the means in the same column with different superscript letters (a-g) Acid and metabolite components produced by the LAB culture support its inhibitory effect against bacterial infections.Lactic acid bacteria's acid has an antibacterial effect on pathogens [2].Bacteria Gram positive also Gram negative generally inhibited in their growth by the antibacterial components generated by LAB [27].Because of a variety of variables, including the generation of lactic acid, which lowers pH, and other inhibitory compounds like bacteriocins, which have the greatest antimicrobial effect, the inhibition's actions changes depending on LAB.[28].The ability of probiotic candidate bacteria to inhibit the growth of pathogens is one of the most important criteria.It has been reported that strain Lactobacillus plantarum-pentosus Mut-7 and Mut-13 (isolated from gatot), Lactobacillus plantarum Dad-13 (isolated dadih), and Lb.acidophilus SNP-2 (material feses of healty infant) were able to inhibit the three pathogenic bacteria i.e Shigella dysenteriae dkt-5, Escherichia coli OK111, and E. coli ST [9].

Lactic acid bacteria's lowering of cholesterol
Results showed that lactic acid bacteria can lower cholesterol in supernatant fluids (Table 2).The amount of cholesterol reduced from the medium in the presence of lactic acid bacterial isolates is between 34%-65%, with the lowest reduce by isolate RS-6 (34%) and higest by RS-9 (65%).The less cholesterol remaining in the media/fluid, it is conceivable that the more cholesterol is contained in the bacterial cells.
When Lactobacillus plantarum KLDS 1.0344, which was isolated from jiaoke, was present, 54.08% of the cholesterol in the supernatant fluids vanished.After being isolated from jiaoke, Lactobacillus plantarum KLDS 1.0344 was able to extract 54.08% of the cholesterol from the supernatant.A portion of the cholesterol (18.57%) precipitated and resolubilized in the washing solutions, a portion (15.69%) remained in the cells, and a portion (19.82%) was not recovered [20].Research conducted by [29], that Lactobacillus casei AG and Lactobacillus casei AP could assimilate cholesterol.A total of 115 strains isolated from fermented foods from Guizhou.Cholesterol reduction test results obtained 56 strains can reduce cholesterol ranged from 7.29 up to 25.66%.Most of the strains could reduce cholesterol by fewer than 15%, and a small percentage could reduce cholesterol by more than 15%.Five strains with a cholesterol elimination rate of more than 20% were isolated from pickled pepper, fermented of glutinous rice flour, cured beef, and Libo sour pork.A rate of 15-20% cholesterol elimination was observed in 13 strains [21].Researchers investigated how lactic acid bacteria, as probiotics, lower cholesterol.Three mechanisms could account for the lactic acid bacteria's capacity to eliminate cholesterol from the medium.The coprecipitation of cholesterol with free bile salts is one of them.It has been reported that it is the deconjugation of bile salts caused by the bile salt hydrolase (BSH) enzyme activity of these strains that allows Lactobacilli to remove cholesterol.The assimilation of cholesterol by lactic acid bacteria cells was another mechanism [30].The incorporation of cholesterol by the strain's cells was solely responsible for the cholesterol revealed in the fragmented-cell solution [24].The third mechanism discovered was the lactic acid bacteria's breakdown of cholesterol [20].
The contribution of this research is related to the environment, sustainability, agricultural activities, and/or agricultural commodities in a broad sense.If lactic acid bacteria are successfully isolated from dengke naniura, and have been tested for their ability to lower cholesterol, it can be utilized for the food processing of agricultural products so that it can be widely consumed by the community.In addition to increasing the diversity of processed agricultural food, it is expected to have a health impact on the Indonesian farming community.

Conclusion
Results lead to the conclusion that as many as 15 isolates of LAB from Dengke naniura can reduce cholesterol in the media.So, it is recommended to conduct in vivo testing using experimental animals.

Table 1 .
Average inhibition zone of LAB culture isolated from dengke naniura

Table 2 .
Lactic acid bacterial ability to remove cholesterol from medium