Phytohormone and amino acid content in golden apple snail (Pomaceae canaliculata L.) extract which fermented at several effective microorganism 4 (EM4) concentrations

This study aims to determine the phytohormone and amino acid content of golden apple snail (GAS) extract fermented using several concentrations of EM 4 Active. GAS were taken from rice fields in Tegalgede Village, Jember. Analysis of phytohormone and amino acid content using LCMS at Bioscience laboratory, Politeknik Negeri Jember. EM4 was activated for 7 days using 5% sugar with distilled water as a solvent. The active EM4 results were taken in several concentrations: 0, 5, 10, 15, 20 and 25% and fermented in GAS extract for 14 days. This research was designed using CRD non-factorial design, consisting of 6 Active EM4 concentrations and repeated 4 times. The results of the research showed that there was the phytohormone IAA (Indole Acetic Acid) with the highest concentration of 0.838% in GAS extract fermented using 25% active EM4, while fermentation without EM4 (0%) contained only 0.641% IAA. Other analysis results showed that 12 types of amino acids were detected, namely arginine, tyrosine, alanine, cysteine, glutamic acid, methionine, glycine, leucine-isoleucine, aspartic acid, proline, threonine and valine. The analysis results showed that the 0% (control) treatment had a higher average amino acid value than fermentation using EM4.


Introduction
Golden Apple Snail (GAS) (Pomaceae canaliculata L.) is freshwater snail from the Ampullariidae tribe originating from the South American region [1], and entered and spread throughout Indonesia around the 1980s [2].This snail is known as an invasive pest.Can damage rice plants with damage levels reaching 10-40%.One life cycle is also relatively fast, namely between 2 -2.5 months and its life span can reach 3 years [3].The reproductive power of GAS is very high with the number of eggs reaching ± 8,700 eggs per season [4].
Apart from the impact of crop damage by GAS, the main problem often experienced by farmers is the scarcity and increase in prices of inorganic fertilizers.Apart from that, the increasing use of inorganic fertilizer doses not only has an impact on increasing production costs but also on soil damage and environmental pollution.Inorganic fertilizers that are used continuously in excessive doses will damage the physical, chemical and biological properties of the soil and the residues will also endanger human health.Currently, people are increasingly aware that agricultural products must also be guaranteed to be safe for consumption and supported by an environmentally friendly cultivation system.To overcome this, one solution is to use organic fertilizer.Apart from being environmentally friendly, using organic fertilizer will increase soil fertility.
On the other hand, the potential of GAS can be an innovation in the world of agriculture if processed properly.Apart from that, it can be a solution to reduce excessive use of inorganic fertilizers.GAS can be processed into liquid organic fertilizer to increase plant growth.Several previous studies also stated that GAS were used as liquid organic fertilizer [5], [6].Apart from that, GAS can also be used as bioactivators or local microorganisms (MOL) to stimulate plant growth [7]- [9].One of the bacteria in GAS is also a phosphate-solubilizing bacterium that produces the phytohormone IAA, a hormone from the Auxin class that stimulates plant growth [10].GAS is also an alternative source of food because it has nutritional content.Every 100 grams of golden snail contains 64 kcal, 12 grams of protein, 1 grams of fat, 2 grams of carbohydrates, and other nutritional content such as iron, phosphorus, calcium [3].
Because of the relatively high protein content, of course this also underlies this research that there are amino acids that can stimulate plant growth [11].These amino acids are the result of the degradation of protein compounds contained in GAS and become simpler compounds through an enzymatic hydrolysis process.This process is a protein breakdown process with the help of protease enzymes.Prosthetic enzymes can be produced from microorganisms such as Lactobacillus sp.[12] which can be obtained in Effective Microorganisms 4 (EM4).The protein hydrolysis process by EM4 is influenced by optimal environmental conditions including temperature and EM4 concentration to support bacterial growth.Therefore, this study aims to determine the phytohormone and amino acid content of GAS extract fermented using several concentrations of activated EM 4. The results of this research can provide basic information about the content of GAS as a basic ingredient for environmentally friendly plant growth-promoting biostimulant products.

Method
This research was carried out starting in August to September 2023 at the Bioscience Laboratory, Politeknik Negeri Jember.GAS were obtained from rice fields in Tegal Gede Village, Sumbersari District, Jember Regency, Indonesia.Altitude the location is at 110 meters above sea level at coordinates 8 o 09'01"S 113 o 43'48"E.Fermentation ingredients include GAS shell and meat, auqadest, white sugar, EM4.As well as fermentation tools including meat grinders, fermentation bottles, measuring bottles, pH meters, digital scales, writing instruments.

Activation of EM4 and fermentation of GAS
This test uses several concentrations of EM4 which have been activated by microorganisms.Activation of microorganisms using EM4 5% + 5% sugar solution with distilled water and fermented under anaerobic conditions at room temperature (25°C-30°C) for 7 days [13].The results of active EM4 were taken in several concentrations to be tested, including: 0%, 5%, 10%, 15%, 20% and 25% and fermented with GAS extract under anaerobic conditions for 14 days [14], [15].GAS Extract is obtained from the process of grinding and pulverizing the meat and shell of fresh GAS.

Data analysis and observation parameters
Analysis of phytohormone and amino acid content using the Liquid chromatography-mass spectrometry (LCMS) method.This research used a Non-factorial Completely Randomized Design.The factors tested were 6 Active EM4 concentration levels including: 0%, 5%, 10%, 15%, 20%, 25% and repeated 4 times.Several concentrations of active EM4 have been tested by previous researchers [13], [14], [15].ANOVA data analysis and Duncan's 5% follow-up test using SPSS 20.0 Observation parameters included the presence of the phytohormones auxin, gibberellins and cytokinins, as well as the type and concentration of amino acids.The best treatment was determined based on the results of Duncan's further tests and based on the highest levels of phytohormones and amino acids from the results of hydrolysis using several concentrations of EM4.

Phytohormone content
The analysis results show that in the Golden Apple Snail (GAS) extract there is only the phytohormone IAA (Indole-3-Acetid Acid) which belongs to the Auxin group.In this test, the hormones cytokinin and gibberellin were not detected.The test results of several active EM4 concentrations in the fermentation of GAS extract are shown in the following image.The data in Figure 1.shows that there was a significant increase in IAA concentration in GAS fermentation using Active EM4.In the control treatment (0%), where fermentation only used a 5% sugar solution, there was 0.641% IAA.The highest increase in the amount of IAA reaching 0.838% was produced from fermentation using 25% active EM4.
Several researchers have suggested that leaf-eating insects can release the hormones auxin and cytokinin [16], [17] .Apart from that, the hormone Salicic Acid was found in the mucus of the mollusc D. Reticulatum [18].However, the presence of IAA in GAS is not yet known for certain whether the snails can synthesize IAA themselves or whether it comes from bacteria that are in symbiosis with GAS.
The linear increase in IAA concentration detected in this study (Figure 1) comes from an increase in the concentration of active EM4.Because EM4 is a bioactivator that contains microorganisms such as Pseoudomonas flourescens, Bacillus sp., Aeromonas sp., and Aspergillus sp.shows its activity as a phosphate solubilizer and produces the hormone IAA [19]- [21].Apart from that, the microorganisms in GAS are thought to increase the amount of IAA.Previous researchers stated that the golden snail contains Pseudomonas flourescens bacteria [22].These bacteria are able to produce IAA and also act as phosphate solvents [23].

Amino Acid content
The analysis results of the content, type and concentration of amino acids of GAS extract are listed in Table 1.Note: Numbers followed by the same letter in the same column are not significantly different based on the 5% DMRT test.

4
Amino Acid Types and Concentrations % (V/V) Golden Apple Snail (GAS) extract was hydrolyzed using EM4 anaerobically for 14 days.Protein hydrolysis is the process of breaking the peptide bonds that connect two amino acids.In this reaction, the protease enzyme as a catalyst together with water breaks down proteins into amino acids [24].The use of EM4 which contains bacteria of lactic acid, bacteria of photosynthetic, yeast is able to produce additional protease enzymes in the hydrolysis of proteins into amino acids.
The data in Table 1 shows that several active EM4 concentrations tested had a significant effect on each amino acid concentration.In the control treatment (0%) the average concentration of all amino acids detected had the highest value compared to other treatments.It can also be seen that the active EM4 concentration of 25% has the lowest average value of amino acids.
Increasing the concentration of EM4 given will of course increase the number of microorganisms and also have an impact on increasing the need for microorganisms to continue to metabolize.The bacteria's need for free amino acids is thought to have caused a decrease in the amino acid concentration detected after 14 days of fermentation, as in Figure 2. It is known that in the enzymatic hydrolysis process, lactic acid bacteria are proteolytic bacteria which are capable of producing protease enzymes, as protein breakers, but for the growth of microorganisms, they need nitrogen and free amino acids as food [12].In addition, a decrease in amino acid levels in GAS extract can occur during the storage process.As in previous research, there was a decrease in protein and amino acid levels in GAS extract after the 7th day of fermentation and decreased further until the 13th day [25].However, in this study, the decrease in amino acid levels in GAS extract at a concentration of 25% EM4 was followed by an increase in IAA level (Figure 1).It is suspected that EM4 microorganisms utilize free amino acids and bacteria as their food source and there is an increase in metabolism, which has implications for the increase in IAA isolates.In previous research, it was known that the longer the incubation time, the more IAA production increased, this was due to adequate nutrition, especially amino acids during bacterial growth.[26].

Conclusions
From the results of this research, it can be concluded that Golden Apple Snail (GAS) can be processed into a biostimulant to stimulate plant growth because there is the phytohormone IAA (Indole Acetic Acid) in the GAS extract, both in fermentation with only sugar (0% control treatment) and fermentation with active EM4 for 14 days.However, the highest IAA concentration was 0.838% in the GAS extract fermented using 25% active EM4.Other analysis results showed that 12 types of amino acids were detected, namely arginine, tyrosine, alanine, cysteine, glutamic acid, methionine, glycine, leucine-isoleucine, aspartic acid, proline, threonine and valine.The 0% control treatment actually had a higher average amino acid value compared to fermentation using EM4 for 14 days.For the sustainability of this research, we will further test this biostimulant on plant growth and test its effectiveness when compared to inorganic chemical fertilizers.

Figure 1 .
Figure 1.IAA content resulting from fermentation of GAS at several active EM4 concentrations aged 14 days

Table 1 .
Amino acid content resulting from fermentation of GAS at several active EM4 concentrations aged 14 days