Effectiveness of LAB filtrate of shrimp pastes products on quality characteristics of fresh stingray (Dasyatis kuhlii)

Stingray (Dasyatis kuhlii) is a type of by-catch fish that is widely used as a product with economic value, but the handling on board is not paid attention to so that when it is landed the condition of the fish is not fresh. This study aims to determine the effect of lactic acid bacteria (LAB) of shrimp paste products in maintaining the quality characteristics of stingrays by chemical testing, namely pH and TVBN, and microbiological testing, namely TPC. This research was conducted from August to October 2022. In this study, BAL was used which was isolated from shrimp paste and stored for 7 days at a temperature of 25-27 °C and the observation time was on the 2nd, 4th, and 7th day. The results of the pH test showed varied results. 7.99 – 9.17. TVBN test results in 627.33 mgN/100 to 1052.16 mgN/100. The TPC value shows a result of 3.86 x 107 cfu/ml – 6.788 x 108 cfu/ml. The effect of LAB of shrimp paste products in maintaining the quality characteristics of stingrays is less effective at room temperature.


Introduction
Each fish has a different rate and pattern of degradation.After the fish dies, there will be many changes that occur naturally, which lead to a decrease in quality and spoilage as a result of enzymatic and microbiological activities.Dasyatis kuhlii is a type of stingray that is mostly landed at the Belawan Fish Auction Site, which is a by-catch type of fish.By-catch fish species generally lack selling value and are often not brought to the mainland.As fish are not included in the main catch target, handling them on board is notgiven enough attention, so that when they land they are not fresh and only certain parts are taken, such as fins.Good handling of stingrays needs to be done properly.This stingray belongs to the type of elasmobranchii fish, where this type of fish has a fairly varied content of urea in the body.Elasmobranchii fish meat has a urea content of around 2,33% [1] which is easily decomposed so that it can cause a sharp odor of urine.Urea will easily decompose into its derivative compounds, namely NH3 and CO2.
Shrimp paste is a product of fermented fish and shrimp which is processed traditionally which can be used as a natural preservative.In the shrimp paste product there are lactic acid bacteria that can be isolated and produce antibacterial compounds, namely bacteriocins [2].The use of antibacterial agents such as lactic acid bacteria and bacteriocins has a very important and good effect in inhibiting pathogenic bacteria growth.This is because lactic acid is able to lower the pH to low so it will be difficult for pathogenic bacteria to survive, while bacteriocin inhibits energy production and protein biosynthesis in pathogenic bacteria [3].
In the previous study, showed that the shelf life of catfish added with lactic acid bacteria showed a longer shelf life than the control [4].Where to use a safe preservative alternative, namely utilizing Lactic Acid Bacteria (LAB) culture.The potential of lactic acid bacteria as food biopreservative agents is inseparable from their ability to produce bacteriocins [5,6] which are antimicrobial compounds produced by several lactic acid bacteria.It is hoped that this can also be applied as a safe alternative to be applied to food products, especially stingrays.

Research site
This research was conducted from August to October 2022.The shrimp paste sampling was carried out at the shrimp paste processing site in Medan Belawan District, North Sumatra Province.Stingray samples were taken at the Belawan Fish Auction Place, North Sumatra.Sample analysis was carried out at the Research and Technology Laboratory, Faculty of Agriculture, University of North Sumatra, Microbiology Laboratory, Faculty of Mathematics and Natural Sciences, University of North Sumatra, and at UPT Fisheries Product Quality Testing Laboratory Medan.The research method used was an experimental experiment using a factorial Completely Randomized Design (CRD) with two factors given, namely concentration and storage time.The experiment was carried out with two repetitions.The concentration of LAB used was 0% (control without treatment), 30%, 50%, 70%, and 100% with different storage times so that the treatment results were obtained from the concentration of LAB.

Tools and materials
The tools used in this study, among others, for the stingray sampling procedure were scales, styrofoam, and millimeter blocks.The tool used for sampling shrimp paste is a zip lock.The tools used for the preparation of stingray samples were millimeter blocks, analytical scales, and plastic containers.The tools used for shrimp paste sample preparation were analytical scales, Petri dishes, mortal, pastel, and Erlenmeyer.The tools used for making filtrate are test tubes, petri dishes, ose needles, Bunsen, autoclaves, Erlenmeyer, plastic bottles, centrifuges, eppendorf tubes, digital scales, 10 ml dropper pipettes, mortal, oven, measuring cups, hot plate stirrers, incubators, and a micropipette and tip.Tools for testing the degree of acidity (pH) are a pH meter and a 50 mL beaker glass.The tools used for TPC testing were colony counters, petri dishes, test tubes, micropipette and tip, spatula, 200 ml Erlenmeyer, vortex, and bunsen.The tools used for TVB-N testing were a blender, burette, glass funnel, erlenmeyer, beaker, coarse filter paper, measuring flask, a set of steam distillation apparatus, and an analytical balance with an accuracy of 0.0001 g.
The materials used in this study were stingrays (Dasyatis kuhlii) and shrimp paste as research samples.The materials used for the sampling procedure for stingrays and shrimp paste were stationery, cameras, and labels.The materials used for the preparation of stingray and shrimp paste samples were distilled water, tissue, and aluminum foil.The materials used for the preparation of the filtrate are MRS Agar media, MRS Broth, cotton, tissue, labels, aluminum foil, and distilled water.The materials used for the pH test were distilled water, pH 4 buffer, and pH 7 buffer.The materials used for the TPC test were plate count agar (PCA), cling wrap, aluminum foil, and distilled water.The materials used for the TVB-N test were 6% perchloric acid, 20% NaOH, 3% H3BO3, 0.02 N Na2B4O7, silicon anti-foaming, phenolphthalein indicator, Tashiro indicator, and methyl red indicator.

Preparation of de Man Rogosa Sharpe (MRS) broth media
The preparation of MRS Broth media begins by weighing 23.4 grams of MRS Broth medium added with 0.02 gr CaCO3 1%, putting into a beaker glass, dissolved with distilled water to a volume of 450 ml.Then homogenized using a hot plate stirrer until boiling.Next, it was put into the autoclave to be sterilized at 121°C with a pressure of 1 atm and left for fifteen minutes.The media is ready to use for the multiplication of bacteria.

Preparation of de Man Rogosa Sharpe (MRS) agar media
The preparation of MRSA media begins by dissolving 12.4 grams of powdered MRSA in 200 ml of distilled water.Then homogenized using a hot plate stirrer until boiling.Next, it was put into the autoclave to be sterilized for fifteen minutes at 121°C with a pressure of 1 atm.The media is ready to use for the multiplication of bacteria.

Preparation of Plate Count Agar (PCA) media
Blinding of PCA media was started by weighing as much as 9 grams of Plate Count Agar media powder dissolved with distilled water up to 400 ml.The medium was boiled using a hot plate stirrer and sterilized in an autoclave at 121°C for 15 minutes at a pressure above 1 atm.The media is ready to use for the multiplication of bacteria.

Sampling of Stingray (Dasyatis kuhlii)
The stingray sampling method was carried out by purposive sampling.Sixteen stingray samples were taken from the Belawan Fish Auction Place with a length ranging from 29-35 cm and a weight ranging from 800-1000 grams.The number of stingray samples taken was determined by the number of treatments to be tested.The stingray samples that have been taken are then put into a cool box filled with ice to keep them fresh.

Sample preparation of Stingray (Dasyatis kuhlii)
Sample preparation begins with measuring the morphometrics of the stingray, including total length, body width, and body weight.Then the stingrays were washed slowly using running water, then divided into several parts, and cut with a size of 8 x 7 x 1 cm with an average sample weight of 100 grams.

Shrimp paste sampling
The shrimp paste sampling was carried out at the shrimp paste processing site in Medan Belawan District, North Sumatra Province.Sampling is taken from finished products that are ready to be marketed.Sampling was carried out by aseptically placing it in a container and then bringing it to the laboratory with a label.

Shrimp paste sample preparation
The sample preparation procedure is by the modified [7], which begins with removing the shrimp paste sample from an aseptic container.The sample is weighed at 1 gram with an analytical balance.The samples were mashed using mortar and pastel.Fine samples were then prepared to isolate shrimp paste bacteria.

Isolation of LAB from shrimp paste
The procedure for isolating lactic acid bacteria from shrimp paste was carried out with modifications [8].LAB isolated from shrimp paste, then dilution was carried out, namely by sprinkling dilution.Samples were weighed 1 g, crushed, and suspended in distilled water.Dilution using sterile distilled water.Samples were diluted at a dilution level of 10-1 to 10-5.Samples were isolated using the pour plate technique in 20 mL MRS agar which had been added with 1% CaCO3 and given ketoconazole as an antifungal.Then the samples were incubated at 34°C for 48 hours.After that, the growing LAB colonies were observed which were characterized by smooth yellowish-white rounds.Then to obtain pure colonies, LAB colonies were subcultured onto MRS Agar medium for purification of the colonies using the streak method, namely by using a looped needle and then incubating again for 48 hours at 34°C.Furthermore, after 48 hours, the growing LAB colonies were observed again, and the colonies were ready to be used for the next process.

Making lactic acid filtrate
Making lactic acid filtrate through a centrifuge process [9], to separate the supernatant from the sediment, 10 ml of MRS Broth media containing pure LAB isolate was poured into an ependop tube and centrifuged for 30 minutes at 3000 rpm at 27ºC.Then it was filtered using a 0.45 μm millipore membrane.The filtrate from lactic acid bacteria is then stored in bottles.
The concentration of lsctic scid filtrate used to determine the quality of stingray (D. kuhlii) was 0% (control without treatment), 30%, 50%, 70% and 100%.Each concentration is 30 ml each.Determination of the filtrate concentration of 30% was carried out by means of 9 ml of LAB put into a beaker glass which was dissolved with 21 ml of distilled water.Then the 50% concentration of the filtrate was carried out by means of 15 ml of LAB put into a beaker glass which was dissolved with 15 ml of distilled water.Then the filtrate concentration of 70% was carried out by means of 21 ml of LAB put into a beaker glass which was dissolved with 9 ml of distilled water.The concentration of 100% filtrate is carried out by means of 30 ml of LAB put into a beaker glass without dissolving it with distilled water.
2.12.Observation parameters 2.12.1.pH value test.The pH determination was carried out using an electronic pH meter [10].Before the pH meter is used, the cathode tip of the indicator is washed with distilled water and dried using a tissue.Then the pH meter was calibrated with buffers 4 and 7. Furthermore, to determine the pH of the sample, the tip of the indicator cathode was attached to the surface of the sample that had previously been pierced.Wait one minute until the digital numbers stabilize.Then, note the number that appears on the pH meter display.

Test levels of Total Volatile Base Nitrogen (TVB-N).
Analysis of TVB-N is an indicator the quality of a fishery product which is characterized by the total vaporized base and the activity of proteolytic enzymes which further increase the TVB value [11].Determination of TVB-N based on SNI 2354.8:2009.First, extraction, 10 g sample was weighed using a beaker, then 90 ml perchloric acid (PCA) 6% was added, and homogenized using a homogenizer for 2 minutes, then filtered the sample using a coarse filter paper.Second, in distillation, 50 ml of extract is added to the distillation tube.Added a few drops of Phenolphthalein indicator (a colorless solution in an acidic state).Added a few drops of anti-foaming silicone.Mounted distillation tube on steam distillation equipment.Add 10 ml of 20% NaOH (at this stage the mixture is alkaline, marked in red).Prepared an Erlenmeyer container containing 100 ml of 3% H3BO4 and 3-5 drops of Tashiro indicator (purple solution).Steam distillation was carried out for about 10 minutes to obtain 100 ml of distillate so that in the final volume there was approximately 200 ml of green solution.Distillation of the blank solution was carried out by replacing the sample extract with 50 ml of 6% PCA, further processing was the same as the sample.Third, titration, titration was carried out against the sample distillate and blank using 0.02 N HCl solution and the endpoint of the titration was marked by the formation of a purple color again.The calculation of Total Volatile Base Nitrogen is as follows: : sample weight (g); 14.007 : atomic weight of nitrogen; 5 2.12.3.Total Plate Count (TPC) test.Microbiological testing on stingrays was carried out using the TPC calculation method.The TPC test uses the procedure listed in [12], where the total bacterial count analysis is carried out by serial dilution using the pour cup method.Weighed as much as 1 gram of the crushed sample then put it into a sterile test tube and added 9 mL of distilled water, then homogenized it for 2 minutes.This homogeneity is a solution with a 10 -1 dilution.Mixing the sample solution was taken 1 mL and put into a bottle containing 9 mL of distilled water to obtain a sample with a 10-2 dilution, then homogenized.Then, dilutions were made up to 10 -5 dilutions.Next, 1 mL of the last dilution (10 -5 ) was taken and transferred into a petri dish using a sterile pipette.Then put 15 mL of agar media into the petri dish and shake it until the surface is even (pouring method), leave the petri dish until the media cools and hardens.Next, the cup containing the agar and sample solution was put into the incubator at 35°C for 24 hours.Observations were made by counting the bacterial colonies in the petri dish with the formula: Where: N : the number of product colonies (colonies/ml or colonies/g) ∑C : the number of colonies on all plates counted; n1 : number of plates in the first dilution counted; n2 : number of plates in the calculated second dilution; d : the first dilution that is counted.

Data analysis
Research data were analyzed descriptively quantitatively using ANOVA to determine the effect of each treatment.

The pH value of stingray (D. kuhlii) during LAB immersion.
The results of the stingray pH test showed that the pH value at room temperature storage had a pH value that did not differ much between the 2nd day, the 4th day, and the 7th day.This happened presumably due to the long time span of 7 days, where the longer storage time can result in an increase in the pH of a product due to the activity of microorganisms in it in decomposing the product.The pH of a product increases because during storage evaporation of important compounds occurs so the product pH increases, and each microorganism also has a pH range as a growth medium.Some microorganisms can live in good pH conditions for growth, namely 4.0-8.0[13].From research conducted on stingray samples with different concentrations and storage times, it was found that the degree of acidity or pH in each sample was in the range of values, namely 8.155 -8.75, where the pH value was in alkaline conditions.The alkaline pH value greatly affects the growth value of the number of bacterial colonies in the sample because it affects the optimum pH for bacterial growth.In the process of fish spoilage, changes pH play a very large role because they affect the autolysis process and attack bacteria [14].A good pH value for preserved fish is between 2.0-5.5 while a good pH for microorganism growth media is between 6.0-8 [15].

The value of TVB-N of stingray (D. kuhlii) during LAB immersion
The length of time the storage is carried out on the stingray samples affects the chemical content in the fish's body.In addition to protein content, stingrays also contain high water content.100 grams of stingray contains a water content of 79.10% [16], which with high water content in the fish's body, will affect the speed of the process of decomposition in the fish's body which also affects the length of storage of fish.stingray.The influence of water content is very important in determining the durability of a food ingredient because water content affects physical properties, chemical properties, and spoilage by microorganisms [17,18].The water content contained in fish is around 70-80% of the weight of the meat [19].The high water content will facilitate the growth and development of spoilage microbes, besides that the protein in the fish body will be easily damaged biologically and chemically, and the surrounding environmental conditions affect the type of microbes that grow.This is caused by several things including prolonged storage, temperature, pH, humidity, water content, and other specific conditions [13,20].The different levels of TVB-N in each sample during observation were influenced by the number of surviving bacteria so the results of TVB were also different because of the bacterial metabolism in the form.In addition, it is also influenced by storage conditions at room temperature, where at 25ºC storage, it is less able to suppress the production of bacterial numbers and also causes an increase in the pH value.When the pH increases to alkaline, the number of bacteria also increases and causes the working processes of bacteria and enzymes to increase, so that the rate of protein breakdown in stingrays increases.TVB is the result of protein decomposition by bacterial and enzyme activity, the breakdown of protein can produce 95% of ammonia and CO2, besides that it is a direct result of the breakdown of protein into total N non-protein fish body into a base with an alkaline pH [21].The results of protein breakdown are volatile and give off a bad smell, such as mercaptans, phenol, cresol, indole, and skatol, including ammonia, trimethylamine, H2S, dimethylamine, and other nitrogenous bases which are the work of bacteria and autolytic enzymes during the decomposition process [22].

The value of the number of bacterial colonies of stingray (D. kuhlii) during lactic acid filtrate immersion
The number of bacteria on the second day of storage was 2.31 x 10 8 cfu/ml, at this stage, the bacteria were just starting to adapt to the new environment.On the 4th day, it showed that the bacterial colonies experienced a significant increase with an average at each concentration of 4.60 x 10 8 cfu/ml at this stage the bacteria had experienced growth and division, while on the 7th day, the number of bacterial colonies experienced a significant increase with an average at each concentration of 2.16 x 10 8 cfu/ml.This is because for 7 days the bacteria undergo metabolism and growth by dividing and for 7 days the bacteria experience a phase of life where it begins with the bacteria adapting to their environment, starting to divide until they die.Bacteria have four life phases, namely the lag phase, where cell changes begin to occur in bacteria causing the cells to become active because the microbial population is experiencing activity, the log phase where cells begin to divide and enter the growth phase, at this stage the most active cell reproduction, the stationary phase where the growth rate slows down, and the death phase where the number of bacterial cells that die is more than the cells that are formed [23].

The value of the number of bacterial colonies of stingray (D. kuhlii) during LAB immersion.
Growth in the number of bacterial colonies is caused by the length of storage time, storage conditions at room temperature, cleanliness during the sample preparation process, and others.The longer the storage time, it will affect the process of the bacteria metabolizing and dividing in the sample, but it also depends on the number of nutrients that can be decomposed in the sample.The storage conditions which are affected by temperature and pH also affect the survival of bacteria.When stored at room temperature around 23-30ºC, this temperature includes the ideal temperature for bacteria to live, so that the growth of bacteria is much better [24].The TPC value varies depending on various factors including the type of treatment, temperature, time, and test method [25].Bacterial growth can also be affected by the product storage period, process preparation, and the application of sanitation and hygiene that are not by standards.This microbial population comes from bacteria that multiply the longer the storage time, another thing that can also happen is unclean equipment.

Conclusion
The results of research on the effectiveness of LAB of shrimp paste products on the quality characteristics of fresh stingray (Dasyatis kuhlii) from the Belawan fish auction several conclusions, the application of LAB in maintaining the chemical quality in the pH test obtained a P-value of 0.747, where P ≥ 0.05 which means it has no significant effect, but in the TVBN test obtained a P-value of 0.000,

3
HCl solution in sample titration; Vb : volume of HCl solution in blank titration; N : normality of HCl solution; 2 : dilution factor.W

Figure 2 .
Figure 2. The value of TVB-N of stingray (D. kuhlii) during LAB immersion.