extfieldsofstudy: Chemistry Medicine

text:
Effect of Marine Bacteria and Ulvan on the Activity of Antioxidant Defense Enzymes and the Bio-Protection of Papaya Fruit against Colletotrichum gloeosporioides

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases in papaya fruit. Its control has been achieved with synthetic fungicides, but the application of marine bacteria and the sulphated polysaccharide ulvan (structural description: β[1,4]-D-GlcA-α[1,4]-L-Rha 3 sulfate, β[1,4]-L-IdoA-α[1,4]-L-Rha 3 sulfate, β[1,4]-D-Xyl-α[1,4]-L-Rha 3 sulfate, and β[1,4]-D-Xyl 2-sulfate-α[1,4]-L-Rha 3 sulfate) from Ulva sp. can be an alternative in the use of agrochemicals. Thus, the objective of this study was to assess the effect in vitro and in vivo of two marine bacteria, Stenotrophomonas rhizophila and Bacillus amyloliquefaciens, and ulvan in papaya fruit’s bio-protection against C. gloeosporioides. The capacity of marine bacteria to inhibit mycelial growth and phytopathogen spore germination in vitro through volatile organic compounds (VOCs) and carbohydrate competition was evaluated. Fruit was inoculated with bacteria, ulvan, and C. gloeosporioides and incubated at 25 °C and 90% of relative humidity (RH) for seven days. Disease incidence (%), lesion diameter (mm), and antioxidant defense enzyme activity (such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were quantified. In vitro, C. gloeosporioides was inhibited by S. rhizophila and B. amyloliquefaciens. In vivo, disease incidence and the lesion diameter of anthracnose on papaya fruit were significantly reduced by marine bacteria and ulvan. Antioxidant defense enzyme activity played an important role in fruit bio-protection against C. gloeosporioides. The application of marine bacteria and ulvan can be an alternative in the sustainable postharvest management of papaya.

Introduction
Worldwide, the loss of fruit at postharvest caused by phytopathogens is estimated to reach 50% of total production [1]. Papaya is a fruit with high demand in the world market; however, due to its water content, it is susceptible to fungal diseases, of which anthracnose is one of the main diseases causing economic losses [2]. The fungus Colletotrichum gloeosporioides is the causal agent of anthracnose, occupying eighth place in the classification of important plant phytopathogens [3,4].
Synthetic fungicides constitute the main method of controlling anthracnose; nevertheless, the use of these agrochemicals generates resistance to phytopathogens, besides the adverse effects on the environment and human and animal health [5]. The presence of synthetic fungicide residuals on food has led to the search for alternatives to control phytopathogens, where the use of microorganisms as 2.5.2. Inhibition of C. gloeosporioides for VOCs The experimental assay was based on the mouth-to-mouth method proposed by Rouissi et al. [38]. Petri dishes with tryptic soy agar (TSA, Difco, BD, Franklin Lakes, NJ, USA) were inoculated in the center with a fungus plug from a 7-day fungus culture in PDA. At the same time, 30 µL of B. amyloliquefaciens and S. rhizophila (1 × 10 6 cells/mL) were individually streaked in other dishes containing the same media. The plate was placed mouth-to-mouth, sealed (parafilm), and incubated at 25 • C for seven days. The growth diameter of C. gloeosporioides was quantified (mm), and the radial growth reduction was calculated with the equation, I (%) = DC-DT/DC × 100, where I% = mycelium growth inhibition in percentage, DC = mycelium measured in control treatment, and DT = mycelium diameter in the presence of the marine bacterium treatment. The control treatment was represented by Petri dishes inoculated only with C. gloeosporioides. Ten replicates were used per treatment, and the experiment was repeated twice.

Competition for Carbohydrates
Starting from fruit at commercial maturity, a concentrate of papaya var. Maradol was made and diluted 1:10 with distilled water and then sterilized in an autoclave at 120 • C for 15 min; after that, 50 mL of the sterile papaya juice medium (SPJ) were placed in Erlenmeyer flasks and inoculated with 500 µL of a suspension of each marine bacteria (1 × 10 6 cell/mL) and 500 µL of a suspension of C. gloeosporioides (1 × 10 4 spores/mL). The Erlenmeyer flasks were incubated at 25 • C and 125 rpm for 24 h; 5-mL aliquots were taken to determine total carbohydrates (anthrone method [39]), sucrose (3,5-Dinitrosalicylic acid (DNS) method [40]), glucose (glucose oxidase-phenol and 4 aminophenazone GOD/PAP [41]), and fructose (tryptamine hydrochloride [42]). The percentage of germinated C. gloeosporioides spores was calculated in 100 spores. Ten replicates were used per treatment, and the experiment was repeated twice.
2.6. Effect of B. amyloliquefaciens, S. rhizophila, and Ulvan on the Control of Papaya Fruit Anthracnose Papaya var. Maradol fruit at commercial maturity was disinfected with sodium hypochlorite at 2% for three minutes, then washed with sterile distilled water and dried in a laminar air flow bench for two hours. Three lesions (3 mm in depth each) were made in each fruit with a sterile needle. Subsequently, each wound was inoculated with 20 µL of each bacterium (1 × 10 6 cell/mL) and ulvan. They were left to dry for two hours, and then each wound was inoculated with 20 µL of a fungus spore suspension (1 × 10 4 spores/mL). A fruit group was inoculated with C. gloeosporioides, and treated with the synthetic fungicide benomyl at a concentration of 1000 ppm. Another group was inoculated only with the phytopathogen. The fruit was placed in sterile plastic containers at 25 • C and 90% relative humidity (RH) for seven days. The disease incidence (%) and lesion diameter (mm) were quantified. Ten replicates were used per treatment, and the experiment was repeated twice.

Sample Collection
After the papaya was inoculated with marine bacteria, ulvan, and/or C. gloeosporioides, 1 g of fruit tissues at 6, 12, and 24 h was collected. Samples were deposited in Eppendorf flasks, adding 2 mL of phosphate buffer (pH 7.8), and the tissues was homogenized with a Polytron tissue homogenizer (Brinkman Instruments) for 60 s. Subsequently, they were centrifuged at 8000 rpm at 5 • C for 15 min. After that, the supernatant was recovered, placed in Eppendorf flasks, and stored at −40 • C for subsequent analysis. Catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities were expressed in units (U) per gram of protein (U/g protein). The protein content was quantified by the method described in [43], using bovine serum albumin (BSA) as a standard. Ten replicates were used per treatment, and the experiment was repeated twice.

Catalase (CAT) Activity
Catalase activity was quantified in 96-well microplates; 10 µL of phosphate buffer of 100 nM (pH 7.0) and 20 µL of the supernatant were deposited in each well and incubated for six minutes. After that, 20 µL of H 2 O 2 (8.82 M) were added and incubated at room temperature for 20 min. A unit of CAT was defined as the decomposition of 1 µmol of H 2 O 2 per min. The samples were quantified in a spectrophotometer at an absorbance of 240 nm [44].

Superoxide Dismutase (SOD) Activity
The SOD activity was quantified at pH 7.8 in Tris-HCl 0.2 M and sodium carbonate 50 Mm (pH 10), adding 20 µL of the supernatant and 20 µL of xanthine/xanthine oxidase. The superoxide generated by the xanthine/xanthine oxidase was detected by controlling the reduction of nitro blue tetrazolium (NAT) and determined in a spectrophotometer at an absorbance of 505 nm. One unit of SOD was defined as the amount of enzyme that causes an inhibition of 50% in the reduction of NAT [45].

Peroxidase (POD) Activity
In 96-well plates, 10 µL of the supernatant and 50 µL guaiacol solution (100 mM of sodium phosphate, pH 6.4, and guaiacol 8 mM) were placed and incubated at 30 • C for 5 min. One unit of POD was defined as an increase of one unit of absorbance per minute under the conditions of the assay. Samples were quantified in a spectrophotometer at an absorbance of 460 nm [46].

Statistical Analysis
Data were processed by a one-way analysis of variance (ANOVA) and Tukey's test with a significance level of 5%, using the software STATISTICA (version 8.0.360.0 StatSoft Inc., Tulsa, USA) for Windows.

Inhibition of C. gloeosporioides Spore Germination by Ulvan
Ulvan did not affect the germination of the phytopathogen fungus spores in any of the time intervals assessed (6, 12, 18, and 24 h). At the end of the experiment, C. gloeosporioides reached 82% of germinated spores, and phytopathogen fungus and ulvan treatment was 83%; thus, no significant differences were found between treatments with and without ulvan + C. gloeosporioides.

In Vitro Antifungal Activity of Marine Bacteria to Phytopathogen Fungus
A significant inhibition was observed in the mycelial growth of C. gloeosporioides by the effect of S. rhizophila, B. amyloliquefaciens, and the synthetic fungicide ( Figure 1A). C. gloeosporioides reduced its growth in 72% by the antagonism of S. rhizophila. The marine bacteria were more efficient in terms of the mycelial growth of the phytopathogen fungus compared with the synthetic fungicide.

Statistical Analysis
Data were processed by a one-way analysis of variance (ANOVA) and Tukey's test with a significance level of 5%, using the software STATISTICA (version 8.0.360.0 StatSoft Inc., Tulsa, USA) for Windows.

Inhibition of C. gloeosporioides Spore Germination by Ulvan
Ulvan did not affect the germination of the phytopathogen fungus spores in any of the time intervals assessed (6, 12, 18, and 24 h). At the end of the experiment, C. gloeosporioides reached 82% of germinated spores, and phytopathogen fungus and ulvan treatment was 83%; thus, no significant differences were found between treatments with and without ulvan + C. gloeosporioides.

In Vitro Antifungal Activity of Marine Bacteria to Phytopathogen Fungus
A significant inhibition was observed in the mycelial growth of C. gloeosporioides by the effect of S. rhizophila, B. amyloliquefaciens, and the synthetic fungicide ( Figure 1A). C. gloeosporioides reduced its growth in 72% by the antagonism of S. rhizophila. The marine bacteria were more efficient in terms of the mycelial growth of the phytopathogen fungus compared with the synthetic fungicide.

Inhibition of C. gloeosporioides by VOCs
S. rhizophila and B. amyloliquefaciens inhibited the mycelial growth of C. gloeosporioides significantly through VOCs ( Figure 1B). The greatest growth inhibition (65%) of the phytopathogen fungus was achieved by the marine bacteria S. rhizophila.

Carbohydrate Competition between Marine Bacteria and C. gloeosporioides
Sucrose, glucose, and fructose decreased rapidly in content in the sterile papaya juice medium (SPJ) when inoculated with the marine bacteria and phytopathogen fungus compared with the

Inhibition of C. gloeosporioides by VOCs
S. rhizophila and B. amyloliquefaciens inhibited the mycelial growth of C. gloeosporioides significantly through VOCs ( Figure 1B). The greatest growth inhibition (65%) of the phytopathogen fungus was achieved by the marine bacteria S. rhizophila.

Carbohydrate Competition between Marine Bacteria and C. gloeosporioides
Sucrose, glucose, and fructose decreased rapidly in content in the sterile papaya juice medium (SPJ) when inoculated with the marine bacteria and phytopathogen fungus compared with the medium only with each microorganism (Figure 2). At 24 h, the sucrose content decreased by 81% and fructose by 65% when SPJ was inoculated with B. amyloliquefaciens and C. gloeosporioides. Glucose reduced by 73% in the presence of S. rhizophila and C. gloeosporioides. medium only with each microorganism (Figure 2). At 24 h, the sucrose content decreased by 81% and fructose by 65% when SPJ was inoculated with B. amyloliquefaciens and C. gloeosporioides. Glucose reduced by 73% in the presence of S. rhizophila and C. gloeosporioides. The spore germination inhibition of C. gloeosporioides by S. rhizophila and B. amyloliquefaciens was quantified at 73% and 65%, respectively. The treatment with only the phytopathogen fungus showed 94% of spore germination at 24 h of inoculation of the SPJ medium.

Bio-Protection of Papaya Fruit Inoculated with Marine Bacteria and Ulvan to Anthracnose
The fruit inoculated with the phytopathogen fungus, marine bacteria, ulvan, and synthetic fungicide showed significantly lower levels of incidence and lesion diameter (mm) of anthracnose compared with the fruit with only C. gloeosporioides (Figure 3). A reduction of 80% was quantified in incidence and 97% in lesion diameter (mm) in fruit inoculated with the phytopathogen fungus and S. rhizophila. The application of marine bacteria and ulvan on fruit was more efficient in the control group of anthracnose than in the treatment with synthetic fungicide.

Antioxidant Defense Enzyme Activity in Papaya Fruit Inoculated with Marine Bacteria and Ulvan
The SOD activity showed the highest levels at 6, 12, and 24 h after fruit inoculation with marine bacteria and ulvan, and the phytopathogen fungus showed the highest level in relation to the fruit without microorganisms and ulvan (Figure 4). At 6 h, the highest SOD values were observed in fruit with C. gloeosporioides and B. amyloliquefaciens; at 12 h, the treatment with the phytopathogen fungus and S. rhizophila; and at 24 h, the highest values were observed in the fruit with C. gloeosporioides and ulvan. The spore germination inhibition of C. gloeosporioides by S. rhizophila and B. amyloliquefaciens was quantified at 73% and 65%, respectively. The treatment with only the phytopathogen fungus showed 94% of spore germination at 24 h of inoculation of the SPJ medium.

Bio-Protection of Papaya Fruit Inoculated with Marine Bacteria and Ulvan to Anthracnose
The fruit inoculated with the phytopathogen fungus, marine bacteria, ulvan, and synthetic fungicide showed significantly lower levels of incidence and lesion diameter (mm) of anthracnose compared with the fruit with only C. gloeosporioides (Figure 3). A reduction of 80% was quantified in incidence and 97% in lesion diameter (mm) in fruit inoculated with the phytopathogen fungus and S. rhizophila. The application of marine bacteria and ulvan on fruit was more efficient in the control group of anthracnose than in the treatment with synthetic fungicide.

Antioxidant Defense Enzyme Activity in Papaya Fruit Inoculated with Marine Bacteria and Ulvan
The SOD activity showed the highest levels at 6, 12, and 24 h after fruit inoculation with marine bacteria and ulvan, and the phytopathogen fungus showed the highest level in relation to the fruit without microorganisms and ulvan (Figure 4). At 6 h, the highest SOD values were observed in fruit with C. gloeosporioides and B. amyloliquefaciens; at 12 h, the treatment with the phytopathogen fungus and S. rhizophila; and at 24 h, the highest values were observed in the fruit with C. gloeosporioides and ulvan.
The CAT activity showed the highest levels in relation to fruit without microorganisms and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 5). At 6 and 12 h, the highest CAT values were observed in fruit with C. gloeosporioides and S. rhizophila, and at 24 h, the highest values were observed for treatments with the phytopathogen and each marine bacterium and ulvan.   The CAT activity showed the highest levels in relation to fruit without microorganisms and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 5). At 6 and 12 h, the highest CAT values were observed in fruit with C. gloeosporioides   The CAT activity showed the highest levels in relation to fruit without microorganisms and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 5). At 6 and 12 h, the highest CAT values were observed in fruit with C. gloeosporioides and S. rhizophila, and at 24 h, the highest values were observed for treatments with the phytopathogen and each marine bacterium and ulvan. The POD activity showed the highest levels in relation to those without microorganism and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 6). At 6 h, the highest POD values were observed in fruit with C. gloeosporioides and ulvan; at 12 h, this was true for the treatment with the phytopathogen fungus and B. amyloliquefaciens; and at 24 h, the highest values were observed for fruit with C. gloeosporioides and ulvan.

Discussion
The biological control of postharvest diseases in fruit using antagonistic microorganisms and ulvan as resistance inductors in plants has been an efficient alternative to the use of synthetic fungicides [32,47].
The marine bacteria S. rhizophila and B. amyloliquefaciens inhibited in vitro mycelial growth of C. gloeosporioides. Particularly, the in vitro antagonism of different phytopathogens by Stenotrophomonas The POD activity showed the highest levels in relation to those without microorganism and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 6). At 6 h, the highest POD values were observed in fruit with C. gloeosporioides and ulvan; at 12 h, this was true for the treatment with the phytopathogen fungus and B. amyloliquefaciens; and at 24 h, the highest values were observed for fruit with C. gloeosporioides and ulvan. and S. rhizophila, and at 24 h, the highest values were observed for treatments with the phytopathogen and each marine bacterium and ulvan. The POD activity showed the highest levels in relation to those without microorganism and ulvan at 6, 12, and 24 h after fruit was inoculated with marine bacteria, ulvan, and the phytopathogen fungus ( Figure 6). At 6 h, the highest POD values were observed in fruit with C. gloeosporioides and ulvan; at 12 h, this was true for the treatment with the phytopathogen fungus and B. amyloliquefaciens; and at 24 h, the highest values were observed for fruit with C. gloeosporioides and ulvan.

Discussion
The biological control of postharvest diseases in fruit using antagonistic microorganisms and ulvan as resistance inductors in plants has been an efficient alternative to the use of synthetic fungicides [32,47].
The marine bacteria S. rhizophila and B. amyloliquefaciens inhibited in vitro mycelial growth of C. gloeosporioides. Particularly, the in vitro antagonism of different phytopathogens by Stenotrophomonas

Discussion
The biological control of postharvest diseases in fruit using antagonistic microorganisms and ulvan as resistance inductors in plants has been an efficient alternative to the use of synthetic fungicides [32,47].
The marine bacteria S. rhizophila and B. amyloliquefaciens inhibited in vitro mycelial growth of C. gloeosporioides. Particularly, the in vitro antagonism of different phytopathogens by Stenotrophomonas spp. is related to diverse antagonistic mechanisms that have already been described for some species of this bacterium, of which the production of antimycotic antibiotics, such as maltophilin and xanthobaccins, are macrocyclic lactams that inhibit phytopathogen growth [48][49][50]. A mechanism of S. rhizophila is lytic enzyme production, such as proteases, chitinases, and β-1,3-glucanasas, which eliminate fungi when they break the links of mannoproteins, chitin, and glucan contained in their cell wall [51]. Stenotrophomonas spp. also inhibit phytopathogens by siderophore synthesis (from Greek sideros phoros "iron bearer or carrier"), which are low molecular-weight molecules that act as specific iron chelators, minimizing availability toward other microorganisms that need this essential micronutrient for cellular metabolism, oxygen transport, respiration, and DNA synthesis, among others [52]. The volatile organic compounds produced by Stenotrophomonas spp. as β-phenylethanol showed an antimicrobial effect, altering the plasmatic membrane permeability in fungi and sugar and amino acid transport processes [53].
Bacillus amyloliquefaciens is a bacterium with a high capacity in vitro to control several phytopathogens [54,55]. The inhibition of C. gloeosporioides by this bacillus is related to diverse antagonistic mechanisms, mainly the production of several metabolites, such as 2-propanona and 2-methylpiridine-both VOCs that inhibit fungus growth and stimulate host resistance induction [56][57][58]. Other antimicrobial compounds that this bacillus produces are lytic enzymes, such as β-1,3-glucanases and cellulose, capable of degrading the fungus cell wall [59], antibiotics; polyketides, and cyclic lipopeptides (LPs), mainly from the surfactin, iturin, and fenesin families, which can easily be combined with the lipid layer of the phytopathogen cell membrane, damaging membrane integrity [60][61][62].
In vivo, anthracnose control in papaya fruit by S. rhizophila, B. amyloliquefaciens, and ulvan was more efficient in comparison to the protection exerted by the synthetic fungicide. The antagonistic microorganisms and ulvan were an alternative to the use and decrease in synthetic applications, which impact the environment and human and animal health negatively, and in the generation of phytopathogen resistance [5,32]. S. rhizophila and B. amyloliquefaciens share diverse antagonistic mechanisms (lytic enzyme production, VOCs, antibiotics, siderophores, among others) involved in the biocontrol in vivo of diseases in fruit; however, competition for space and nutrients and the induction of host resistance are two important mechanisms that both bacteria have for phytopathogen biocontrol [36]. In this study, the marine bacteria reduced C. gloeosporioides spore germination because of sucrose, glucose, and fructose, which were consumed rapidly by S. rhizophila and B. amyloliquefaciens, decreasing the content of strong carbon sources for their absorption by the phytopathogen. Carbohydrates are of great importance and are useful for spore germination and start the infection process of C. gloeosporioides to fruit [63].
On the other hand, bacteria and ulvan have been considered to be inductors of plant resistance [64] and a biotechnological control option of phytopathogens of agricultural importance [33]. In this study, S. rhizophila, B. amyloliquefaciens, and ulvan protected papaya fruit against anthracnose through the induction of defense antioxidant enzymes, such as SOD, CAT, and POD, whose activities (U/g protein) were higher in fruit treated with marine bacteria or ulvan in relation to the treatment with only the phytopathogen. Thus, the resistance inductors in plants play an important role in disease reduction [35,36]. The function of defense antioxidant enzymes is to protect the host from reactive oxygen species (ROS), such as superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH − ), caused by the phytopathogens in their infection process [65,66]. ROS can damage nucleic acids, proteins, carbohydrates, and lipids, and affect cell membrane integrity, directly inactivating key cell functions in plants [67]. For plants, the proportion between the generation and elimination of ROS is transcendental in all cells; thus, defense antioxidant enzymes play an important role in intracellular equilibrium. SOD is a detoxifying enzyme that allows the elimination of ROS in the cell [68]. With respect to POD, it is found in all host cells, protecting them against the destructive activity of H 2 O 2 through the oxidation of phenolic and enodiolic co-substrates [69,70]. Finally, CAT is found in aerobic cells and protects them from H 2 O 2 , catalyzing its decomposition in O 2 and H 2 O [71]. Although several studies of bacteria and ulvan exist for defense antioxidant enzyme induction (SOD, CAT, and POD) in plants for their bio-protection against different phytopathogens, such as Alternaria tenuis and Botrytis cinerea in peach [8], B. cinerea in pepper [43], C. gloeosporioides in lychee [72], C. acutatum in loquat [73], Penicillum expansum in apple [35], and C. trifolli in Medicago sp. [33], this is the first report of the marine bacteria S. rhizophila and B. amyloliquefaciens and ulvan as resistance inductors in papaya fruit against anthracnose caused by C. gloeosporioides.

Conclusions
S. rhizophila and B. amyloliquefaciens inhibited the in vitro growth of C. gloeosporioides; additionally, the application of both marine bacteria and ulvan on papaya fruit protected them against anthracnose. The mechanism that participated in fruit bio-protection by the antagonistic bacteria and ulvan was competition for sucrose, glucose, and fructose production by VOCs and defense antioxidant enzyme induction, such as SOD, CAT, and POD. Future studies assessing the efficiency of S. rhizophila, B. amyloliquefaciens, and ulvan at a greater scale should be performed on papaya fruit as an alternative to the use of synthetic fungicides and the sustainable management of agricultural products at postharvest.


extfieldsofstudy: Physics Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Response surface modeling and optimizing conditions for anthocyanins extraction from purple sweet potato (Ipomoea batatas (L.) Lam) grown in Lam Dong province, Vietnam

Anthocyanin is increasingly used as a natural and safe coloring agent. In this paper, the extraction of purple sweet potato anthocyanin (PSPAs) was investigated by using response surface methodology (RSM). Different extraction temperatures of solvent ethanol (60 – 70 °C), duration of extraction (35 - 45 min) and solid-liquid ratios (4:1 - 6:1) were selected in order to extract PSPAs. The highest anthocyanin content of 206.019 mg/L of PSPAs was collected at the solid liquid ratio 6:1, extraction time 39.61 min, and temperature 67.38°C. PSPAs yield detailed significant correlation with high F values, low P values (<0.0001), the determination coefficient (R2=0.9986) and a high desirability 93.5%.

Introduction
Anthocyanins, apart from being recognized as natural plant pigments from the flavonoid family, possess valuable pharmacological properties such as anti-oxidative, anti-inflammatory and antineurodegenerative effects. Despite the ubiquity of the substance in many flowers and fruits of the high plants, it is the purple sweet potato (Ipomoea batatas (L.) Lam) that has attracted attention thanks to its high content of anthocyanins, unique color, nutritional value and health benefits. In particular, multiple physiological functions including antimutagenicity, antihyperglycemic effect and free radicalscavenging activity are exhibited from natural purple sweet potato color (PSPC), commonly extracted from purple sweet potatoes [1,2].
Compared to other variants of sweet potatoes, purple sweet potatoes have high antioxidant activity of PFSP thanks to the high content of anthocyanins in the forms of mono or diacylated cyanidin and peonidin [3]. The analysis of nutraceutical properties of PFSP has revealed that anthocyanins have strong radical scavenging and and antimutagenic activities. In addition, when tested on rats, the substances could also reduce blood pressure and prevent carbon tetrachloride-induced liver injury. Other physiological functions of anthocyanins include anti-inflammatory, antimicrobial, and ultraviolet protection effects [3,4].
Response surface methodology (RSM) is a collection of statistical techniques and empirical model built devised to examine the impacts of two or more variables (input variables) on a response variable using multiple regression and correlation analysis [5][6][7][8][9]. Through a careful experiment design, the technique aims to optimize a response output variable, influenced by several inputs, in order to attain the best system performance. In this study, extraction temperature, extraction time and solid-liquid ratio are identified as the main factors, and are therefore assumed to be correlated with the yield of anthocyanins extracted from purple sweet potatoes. For each variable, three values, coded as -1, 0 and +1, corresponding to low, intermediate and high value, respectively, were designated. The second order polynomial modal, which related to the selected variables, is shown below: Where, Y is a response variable, , , and are the regression coefficients of the intercept, linear, quadratic and interaction terms, respectively. x i and x j are coded values of independent variables affecting the response of Y.

Sample preparation
PSPAs, rich in anthocyanins, were grown in lam Dong, Viet Nam. The harvested potatoes were washed, cut into cubes (3-5mm), dried by microwave 400W for 30 min, pulverized, and the collected powder was stored in dark black bottles in room temperature for further use. Ethanol (C 2 H 5 OH) is purchased from Sigma Aldrich (US).
For extraction parameter study, 15g of purple sweet potato powder was put in the two neck round bottom flask and was extracted by H 2 O, EtOH40 o -EtOH70 o (H 2 O:Ethanol = 6:4 to 3:7, mL/mL) solutions, material/solvent ratio: 4:1 -8:1 (g/mL). The extraction temperature is adjusted to about 30 -70 ( o C) and time of extraction was restricted from 40 to 70 (min). Then, centrifugation took place at 4000 rpm for 15 min. The supernatant was collected and the extract, after being filtered with filter paper, was transferred into plastic bottles for to estimate anthocyanin yield. The pH scanning of supernatant ranges from 400 nm to 700 nm.

Total monometric anthocyanin by the pH-DIFFERENTIAL method
The pH-differential method was used to determine the total monomeric anthocyanin content. Each sample was diluted twice. Sodium acetate (0.4 M) at pH 4.5 and potassium chloride (0.025 M) at pH 1 were used in the first and the second dilution, respectively. At 521nm, absorbance readings of the samples must not exceed 1.2. Following an equilibration period of 15 min, absorbance at 400 and 700 nm of the samples was recorded using a spectrophotometer calibrated with distilled water as the blank. Absorbance was calculated at pH 1.0 and pH 4.5. Calculation of Anthocyanin content (a) follows pHdifferential method as follows: (2) Where: A = (A 521nm -A 700nm ) pH1,0 -(A 521nm -A 700nm ) pH4,5 . a : the total anthocyanin content (mg); A 521 , A 700 : absorbances at 521 and 700 nm respectively; MW: cyanidin-3-glucoside molecular weight (449.2g/mol); DF: dilution factor; ɛ: cyanindin-3-glucoside molar absorptivity (ɛ =26900); l : measurement cell path length; 10 3 : conversion factor from g to mg [10].

Single factor investigation
As mentioned before, four contributing factors to the extraction of anthocyanin were concentration of Ethanol solvent (mol/L), time (min), temperature ( o C) and solvent/material ratio (mL/g). Therefore, select the optimum condition by testing each of the four alternatives in the selected element.

Optimization of RSM
The extraction experiments were performed by Design Design-Expert® software version 11 (DX11) from Stat-Ease Inc., Minneapolis, Minnesota. Based on the results of single factor experiments, three independent variables were identified: ratio solid/solvent (X 1 ) was 4:1 -6:1 (mL/g), temperature (X 2 ) at 60-70 ( o C), and time on the extraction (X 3 ) was chosen 35-45 (min) to evaluation. Table 1 displayed Total anthocyanin contents (TAC) obtained.

Statistical analysis
Following a RMS design planned by Design-Expert software (Version 11.0), triplicate extraction experiments were performed and data that accrued from the experiments was analyzed with the same software.

Single factor investigation
Effects of four individual factors on the anthocyanin yield were summarized in Figure 1. Figure 1(a) showed a rapid rise of anthocyanin content as the concentration of ethanol solution increases (other factors: liquid/solid ratio 5:1, time extraction 40 min, temperature 65 o C). However, rising the concentration of the solution from 60% to 70% caused the anthocyanin content to recede. Similar trend could also be interpreted for the impact of temperature, as demonstrated in Figure 1(b). To be specific, anthocyanin yield steadily increases before picking at 60 o C (other factors: ethanol 60%, time extraction 40 min, liquid/solid ratio 5:1). The reason for this is that the increase in temperature will promote the phase separation of anthocyanin from purple sweet potato. To achieve the highest performance when extracting, the temperature will be chosen at 60 o C. For liquid/solid ratio, Figure 1 (c) illustrated that the peak of anthocyanin content is reached at liquid/solid ratio of 5:1. Other anthocyanin yields associating to other ratios were all significantly lower than such threshold (other factors: ethanol 60%, time extraction 40 min, temperature 65 o C). The general trend for reaction time in Figure 1(d) was that the longer the reaction time, the higher the produced anthocyanins, with the exception of yields in the period from 30min to 40min, where a mild increase in yield took place (other factors: ethanol 60%, temperature 65 o C, liquid/solid ratio 5:1). Herein, concentration of ethanol 60%, extraction time 35-45 min, temperature 60-70 o C and reaction time 30~50 min were considered to be optimal in this RSM design.  Figure 1. Effects of four different factors on the content of extracted anthocyanin.

Optimization of RSM
The results of 20 experimental runs with the RSM model were completed design and shown in Table 2. The results implied that the yield of anthocyanin would be severely changed as survey parameters change (ratio, temperature, time). In the table, No.1~14 were the factorial experiments, and No.15~20 were the central experiments. The anthocyanin content ranges from 181.826 mg/L to 212.585 mg/L. The different between actual values and predicted values was not substantial indicating that the results of the experimental experiments have high accuracy. Table 3 show that the factors interacting with yield anthocyanin with significance level R 2 = 0.9986 and confidence level was 93.5%. Overall, the model is significant as demonstrated by the F-value of 814.97. The probability of the model to obtain such value owing to noise is minimal (0.01%). All model terms are also statistically significant as indicated by low p-values. Insignificance of the lack of fit shows that the model fits well with the data, thus no further specification of the model is required. After estimating model coefficients, the following model is obtained: Y = 212.41 -3.24X 1 + 3.76X 2 + 1.40X 3 + 1.92X 1 X 2 -3.99X 2 X 3 + 3.92X- The model is fit with the response variable and independent variables. Therefore, anthocyanin content of 206.019 mg/L was extracted by the optimum parameters as X 1 = 6:1, X 2 = 67.38 , X 3 = 39.61 in order to collect desirability 93.5%, which was completed by DX11 software. Model suitability is accentuated by three statistics, model F-value, P-value of coefficients and the lack of fit. The Model Fvalue of 918.67 implies that the model is significant as there is only a 0.01% chance that a "Model F-Value" this large could occur due to noise. For p-value of coefficients, the results pointed out that all model terms are significant at 1% confidence level. This signified the unnecessity of inclusion of additional model terms. Insignificance of Lack-of-fit value also indicated reasonable fitness of the data to the resulting model. Table 3. ANOVA data for removal models. Based on the model estimated by the software, three response surface plots of the anthocyanin yields, corresponding to different combinations of A, B and C, were plotted as in Figure 2. The contour plots represented impact intensity of the interaction variables on the yield. According to the model results, all three interaction terms representing combinations of three factors are significant (P<0.05). The liquid-to-solid ratio positively affects anthocyanin yields. This is explained by the fact that material surface was swelled when more solvent was present leading to more anthocyanin yield. Temperature and extraction time also aided in the thermal accumulation through increased microwave energy and in turn destabilized cell walls, releasing higher amount of anthocyanins to the solution.

Source
Sum of Squares From the response surface analysis, optimum process of anthocyanin extraction are identified with the following parameters: temperature 67.38 o C, time 39.61 min, and liquid-solid ratio 6:1. The maximum predicted anthocyanin content was 206.019 mg/L. For convenience purposes, we performed the experiment with following parameters to validate the aforementioned optimum process: temperature 65 o C, time 40 min, and liquid-solid ratio 5:1. The results is showed in the Table 4. Evidently, the difference between the actual yield and the predicted yield was subtle indicating relative accuracy of data produced by the model to the actual experimental results.  2. 3D model about the influence and interaction of Y with a) X 1 and X 2 , b) X 1 and X 3, c) X 2 and X 3 .

Conclusions
The optimal levels of three factors were founded by single factor investigation (liquid/solid ratio, extraction time, reaction temperature). Following the single factor assays, we conducted 20 experiments planned by RSM to optimize of the extraction process of anthocyanin content. The results of surface response methodology revealed that the optimum combination of process variables for response functions was temperature 67.38 o C, time 39.61 min, and liquid-solid ratio 6:1, which showed the highest anthocyanin content of 206.019 mg/L.


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Utilization of Tofu Dregs as Goat Fodder and Utilization of Nano Particles for Skin Care Products

The purpose of this community service is to improve the welfare and income of partners through the use of tofu dregs into animal fodder. Then, it will correlate to the quality of milk which produced by the goat. Finally, this goat’s milk will be processed through nanoparticles become a moisturizer for skin care. The location of community service is dedicated for small businesses in Lamnyong Village, Kuala Syiah sub-district, Banda Aceh. In this activity, a method was introduced tofu dregs could be used as quality food for goats which effects on goat milk were highly nutritious. In addition, nanoparticle processes will be introduced on the process of goat’s milk which can be a moisturizer for skin care. This will be a new product that can be mass produced and marketed by partners and continuous promotion, thus increasing consumer interest in buying, which will increase partner income. The result of this service is the addition of new fodders resulting in better quality milk products, this is evidenced from laboratory results and other results is the creation of a skin care product that functions as a skin moisturizer. Finally, this product can be used to soften the skin and protect the skin from the sun.

Introduction
In order to be healthy and good, everyone needs milk as a source of energy. Milk can be easily found in various types and brands in the market. Foods that are circulating in the market usually come from the results of animal husbandry, sheep, goats, etc. and the results of processing agricultural products such as soybeans. Because of different sources, even though it is equally beneficial for the body, each milk produced from milk content is also different.
Today, the milk production process is managed in a traditional and modern way. In modern milk is produced with the help of technology such as the procurement of high-capacity and sophisticated machines. Although it uses many technologies, there are some businesses which still used traditional milk-squeezing to maintain the nature and freshness of the milk.
Utilization of Goat Milk is widely known for maintaining skin beauty and smoothness. Goat milk contains many vitamins, especially vitamins A and D. These vitamins will make the skin soft but strong. Goat milk in this service contains beta hydroxy acid which makes the dead skin cells peel off and replace them with new skin cells so that the new skin is fresher.
George Dermit, a doctor from Ohio University has conducted research and therapy on Goat Milk for children with skin diseases. From the results of the study, he concluded that goat milk is very good for sufferers of skin diseases such as eczema, zits, and itching. Moreover, the dermatologists from New Zealand also apply this to any country in the world.
Goat Milk which is rich in natural hydroxy beta acid is now often used as a mixture for scrubs. This mixture not only functions as a peeling that erodes dirt and dead skin cells but is able to brighten the skin to look smoother and not scaly. Additionally, the protein content in milk is also useful as a nutrient supply that functions to moisturize while coating the surface of the skin to make it more smooth and supple.
Besides the benefits of goat's milk, through nanoparticle processes, it can produce skin care moisturizers. This process must use the quality goat milk to produce good and healthy products. This moisturizer is useful for various skin problems.

Current Conditions and Partners Problem a. Production Aspect
In the business processing, several obstacles faced by UD. Atjeh Livestock farm in producing milk from the aspect of production, there are: 1) Livestock change because looking for other goats takes a long time and requires a large amount of money. The type of goat that produces quality milk is the result of crossbreed originating from India. This type of goat, it can only be found in Medan and Central Java. It is because of the kingdom in the past, Indian traders came and traded in Indonesia with traders from Batavia, Medan, and Java. They crossbreed their livestock to produce quality milk and there was a crossing which produced a type of crossbreed goat. 2) Quality of human resources in the field of animal husbandry is still limited. Then, the number of workers at the production site is very small. The goat milk business is run by an owner with two workers. However, the milking goat process, there is more labor is needed which is caused business becomes lighter.
3) The amount of production is still limited which make them hard to establish a partnership or become a supplier with other businesses. This causes production to be limited to consumers who only need goat milk to drinks, it cannot reach other aspects such as cheese, yogurt, soap, and so on. 4) Lack of adequate production equipment such as goat milk equipment that uses existing machinery and equipment is limited, such as milk cooking tools that use machines as well, so that group members can more easily produce goat milk, and the value added can be enjoyed by group members.

b. Management Aspect
In business management, the goat milk business group experienced several obstacles, there are: 1) Lack of workers who are experts in their fields in conducting business activities such as feeding and drinking livestock, milking, cleaning cages, and others. 2) Weak in marketing. The lack of market information and skills in marketing products has resulted in the low sales which can be obtained by the goat's milk business, because they market their products without special promotions, only rely on mouth to mouth, a sales system that only relies on customer trust so that one customer will tell the experience of buying products and get health benefits from consuming these products to other prospective customers.

Problem of Partner Priority Problem
We draw several priority issues to be resolved so that the goat milk business group becomes more developed in aspects of milk production and business management. Some of the priority issues are: a. Production Aspect 1) Keep increasing the crossbreed goat quantity to produce quality goat milk.
2) Keep increasing the quality of human resources in the field of animal husbandry and squeezed milk. 3) Keep increasing the production capacity by adding milk machines, to create the faster and more numerous products, and also the profits obtained can increase with the milk produced.
b. Management Aspect 1) Increased number of workers who are skilled in the field of goat farming in managing goat milk business. 2) Increased marketing capacity and capability, in terms of analyzing share expansion and market information. 3) Increased capacity of actors in the business of goats in terms of organization/ management.

Solutions Offered
Regarding the main problems of the partners that have been stated, the solutions offered are: a. Production Aspect From the aspect of production, the solutions offered by the Science and Technology program for society include: 1) Increased capacity for the number of crossbreed dairy goats to produce quality goat milk by collaborating with suppliers of dairy goats. Then, when livestock changes it becomes easier. 2) Increased number of qualified human resources in the field of animal husbandry by employing graduates in the field. 3) Increased goat milk production capacity by increasing the number of dairy goats so that they can collaborate or become suppliers with other businesses. 4) Provision of goat milk milking machines to further accelerate production, which is accompanied by a mentoring program to use the tool. 5) Increased product marketing capacity, so partners can market their products with their own brand and packaging and also make consumers find out about these products.

b. Management Aspect
From the management aspect, the solutions offered from science and technology programs for members of this group include: 1) Training in Human Resource Management, which is the improvement of self-development management expertise, which is related to the production process of goat milk. 2) Increased marketing capacity and capabilities, in terms of expanding market share and market information.

Methodology
The community services program has been conducted include product creativity and capacity building for partner business organizations. Therefore, the application of science and technology is carried out through a comprehensive and multidisciplinary approach. The program must also involve the perpetrators of the goat milk group as beneficiaries of the program. The training method is designed using a participatory approach with reference to the adult learning process, through an approach: Presentation of material equipped with PowerPoint Presentation and simulation, Discussion: limited, group, Practice, and Feedback from business actors in the form of understanding the material provided.
Increased production capacity carried out through two fields includes (1) training in marketing goat milk, (2) procurement of dairy machines and other processing equipment, both of which are accompanied by assistance. Mentoring is done by involving parties who are experts in their fields.
To improve capabilities in the marketing field, partners will be given material related to marketing techniques such as determining target consumers, analyzing the market or consumer needs, product marketing through the internet (web), making brand and product logos, as well as market development and breakthrough techniques market.
Development in the field of human resources is held through training and development of workers' expertise, increased collaboration among group members, and strategies for creating a group environment situation to brainstorm or design thinking in a short time, and provide motivation to the goat milk business group to encourage quality dairy products, beneficial to health and have a high

Processing of Animal Fodder into High Quality of Goat Milk
This community service focuses on how to increase farmers' income with various innovations, especially regarding goat feeding to improve the quality of goat milk so that goat milk produced is of high nutritional value and is favored by the community. There are many things have been discussed together with farmers. In the discussion, there were still many shortcomings that needed to be addressed. Among other things is equipment that is still very lacking in handling goat's milk. In addition, in this program, it tries to market dairy products to more extensive things, such as online systems. In the following picture, a discussion of the work plan of service and discussion in terms of overcoming the problems that have occurred so far. Discussions with Pak Bambang in terms of this service can be seen in Figure 1 below:

Figure 1. Road Map of Community Services
Additionally, there are other innovations that devotees try to do, namely by utilizing goat milk into skin moisturizers. Goat milk can be made into a skin moisturizing product through the nanoparticle system. If this product is finished, then this skin moisturizing product can be bought and sold to the public and it can increase farmers' income. However, to produce a good moisturizing product, go through good goat milk. Quality goat milk can be obtained from good goat fodder. Therefore, one of the food sources that must be considered is tofu dregs. It is the best and most nutritious food for goat.
The disadvantage of tofu dregs is that tofu dregs products cannot be stored for a long time, so every purchase of tofu dregs must be calculated when it can run out, so the tofu dregs are not wasted. Tofu dregs are mixed foods where the main food is grass. Mixed foods are very influential on goat's milk resulting from. Therefore, assistance from community service is very helpful for farmers in developing their business. The table below, assistance provided when visiting a goat farm. Giving out the tofu before serving is that they only buy tofu dregs and then give it to the goat. Then, another innovation which can be applied is to provide better quality food. The method used is by the addition of feedings from before. The table below will illustrate the prior fodder of the business, as it showed in the table: In the implementation of the Grant program, additional food was provided to increase the production and quality of the milk. Giving these foods is mixed with other foods. The types of food additives can be referred in the following table 2: Table 2. Additional Fodders of goat livestock 1 Sago flour Second Quality 2 Coconut Meal The addition of these foods is based on the high protein from the feed. Sago flour is high in energy so it will increase milk production and viscosity of milk. While coconut meal is high in vegetable protein which it can increase the milk production. The availability and continuity are very good for milk production. The amount of food given per day per goat livestock is: a. Feed the concentrate of 3% from the weight of livestock life = 0.9 kh/head/day b. Forage fermented 10% of livestock weight = 3kg/tail/day The results obtained after getting additional food from the program, there are as follows: a. Increasing the amount of goat milk production per day. The previous increase in milk production from 1 liter/head/day to 1.3 liters /head/day. b. Increased levels of milk nutrients that are better than before c. The weight gain of dairy goats for 1 month averages 0.1 kg /head/day The comparison of the analysis of milk quality before and after the addition of food is: After doing analysis in the laboratory, the quality of milk after adding the food ingredients is an increase in milk protein levels, milk fat, and milk lactose. Laboratory testing is carried out after 1 month of observation.
The lack of tools in the process of milking and milk production is also very pronounced in this effort, therefore we need the help of machines to launch this business process. The tools that exist before the programs are: a. Clean water is used to clean the udders of the main livestock b. A clean washcloth is used to clean and dry washed udders c. Portable milking machine d. The power cord as a source of electric current for the appliance e. Glass liters are used for milked milk containers f. Milking milk storage containers g. Milk production logbook h. Stationery for recording i. Gauze for milk filters j. Stainless pan for pasteurization The equipment available is limited, therefore it needs help to add the tools for the business to smoothen again. The assistance of these tools includes: a. Portable Milking machine for milking equipment b. The CMT tool is used for mastitis disease testing in the main livestock c. Milk fat test kits are used to test milk fat levels d. The pH meter is used to see the pH of milk e. Bio-lactation flour is used for additional milk production minerals in the main livestock f. Drums are used to store and ferment tofu dregs mixed with concentrate The provision of feeding assistance and tools can be seen in figure 2 below:

Figure 2. Provision of dedication assistance
All assistance provided has the goal to the small and medium entrepreneurs in order to get better milk production than before so they can increase their income, it can expand their business and the can add more workers. Other analyzes conducted to see the quality of milk are analyzes of the microbial composition. This analysis has been carried out and is still awaiting laboratory results from BPOM and it is expected that in the near future the results will be obtained. Figure 3 below, is the process of mixing food for dairy goats and the process of milking goats. However, after obtaining good quality goat milk, then this service will also process goat milk into skin moisturizing products for skin health through particle nano processes. The manufacture is carried out in a chemical laboratory to produce good products. In addition, these skin products will be examined by dermatologists and chemists to ensure that the process of converting milk into skin moisturizing products is correct. The skin moisturizing product is expected to be a product that is beneficial for skin farmers and can be marketed to the general public which it can improve the visibility of entrepreneurs.

The Processing of Goat Milk into a Skin Care Product
Hypoallergenic moisturizers (Hypo-Allergenic) are moisturizers that do not contain substances that can cause reactions allergies to the skin. This type of moisturizer is suitable for sensitive skin. Hypoallergenic means "minimizing the appearance of allergies". The term Hypo-Allergenic for skin care products means that the product uses as little as possible raw materials that can cause allergic reactions.
The use of Hypo-Allergenic products does not mean 100% free from allergy risks, but the reactions that occur due to some very sensitive raw materials on the skin can be limited to a minimum level. A product needs to be tested to prove that the product does not cause skin irritation or allergies. Products that have been tested by a dermatologist and proven to not cause skin irritation allergies are called To produce Hypo-Allergenic products can be done by removing allergens. In addition, the used of natural ingredients that function as an antioxidant and anti-bacterial, anti-inflammatory and others into skin moisturizers can also produce Hypo-Allergenic moisturizers.

CONCLUSION
From the community service program carried out to get a positive response for farmers and hopefully this collaboration can increase knowledge of farmers in continuing to manage goat's milk in accordance with the knowledge obtained from the service system. Then, the skin moisturizing products that are produced can also be produced in large quantities and can be marketed online and also manually. Dairy products produced in this service, become healthier which can increase the purchasing power of other communities. In addition, effective marketing, such as online, can introduce goat's milk products to be better in marketing, so that communities outside Aceh can buy and order online.
The skin care moisturizing products produced are also very helpful to partners in terms of new product introduction. This moisturizing product is much favored by women, especially those whose skin is often exposed to sunlight. So, what exactly is the superiority of goat's milk compared to other milk. Basically, both goat milk, cow's milk, and camel milk are rich in protein, vitamin A, vitamin D, and other minerals. Goat milk has a higher protein and vitamin A content than cow's milk, as well as smaller fat molecules. Goat's milk is also usually richer, making it suitable for pampering the skin like a soap and milk bath product.


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Salt Stress Triggered Changes in Osmoregulation and Antioxidants in Herbaceous Perennial Inula Plants (Asteraceae)

Global demand to cure ailments is a growing need. Inula genus extensively holds hundreds of species in warmer regions of Europe and Asia. It is being well-known for its phytochemical and pharmacological applications in industry thanks to its anti-inflammatory and antimicrobial interests. However, growth and production of Inula in the cuttingedge industry is commonly influenced by salt stress except for the halophyte species such as the Inula crithmoides. Salt tolerance level by means of changes in osmoregulation and antioxidant systems in an herbaceous perennial Inula plant has been biochemically evaluated here. Both salt stress treatments caused photosynthetic pigments’ degradation, increase in the leaf levels of osmolytes, and induction of oxidative stress indicated by the malondialdehyde (MDA). Higher hydrogen peroxide (H2O2) amount was recorded in high salt concentration than low salt. High salinity caused an increase in ascorbate (ASC) and glutathione (GSH) contents besides target enzymes of Inula leaves. NaCl tolerance of Inula also was found comprehensible through the higher concentrations of proline and to a lesser extent, total soluble sugar. Salt tolerance mechanisms of this rich bioresourse needs to be further studied in detail for herbal medicines in pharma sector. Please cite this paper as follows: Pehlivan, N. and Saruhan Guler, N. (2019). Salt Stress Triggered Changes in Osmoregulation and Antioxidants in Herbaceous Perennial Inula Plants (Asteraceae). Alinteri Journal of Agriculture Sciences, 34(1): 39-46. doi: 10.28955/alinterizbd.541949

Introduction
Many geographic regions in the world has undergone changes due to increasing temperatures and fluttering precipitation regimes subsequently affects all aspects of life on earth. Excessive temperature and drought cause increase in soil salinity and significantly reduces product yield and quality. The effects of drought and temperature can be reduced by irrigation however; it leads to a further reduction in the already diminishing water resources. In any event, agricultural activities are the biggest factor in reducing the fresh water reserves in the world. Furthermore, irrigation increases soil salinity. Considering the fact that the world population continues to grow (UN's estimation is 8.6 billion people by 2030) and arable lands are shrinking, it is evident that, yield should be increased at a high rate for nutritional and feeding purposes, for the reason that abiotic stress conditions pose a serious threat to agriculture. Plants cannot escape the conditions in which they exist and are victims of constantly changing conditions, yet they have evolved by developing biochemical and developmental signal cascades for the sustainability of growth, development and agricultural phytomass re-productivity.
Stress adaptation is ensured by the activation of the salt stress susceptible genes and the synthesis of various functional proteins result in restructuring of the relevant signaling pathways (Shinozaki and Yamaguchi-Shinozaki 2007). Reactive oxygen species (ROS) are generated in plants exposed to salt stress and are produced continuously. ROS is highly reactive and its overproduction has been shown to adversely affect membrane potential and the protection of biological macromolecules such as proteins, carbohydrates, lipids and DNA which ultimately leads to oxidative damage (Demiral and Turkan 2005).
Plants develop enzymatic and non-enzymatic defense systems to keep ROS at an optimal level that will not cause damage to the cell or remove redundant ROS from the cell and protect cells from further oxidative injury.
Ascorbic acid and glutathione are classified as nonenzymatic antioxidants, while primary enzymatic ones are superoxide dismutase (SOD), glutathione/ascorbate peroxidase (APX, GPX) and glutathione reductase (GR) (Foyer and Noctor 2009). Glutathione (L-gamma-Glutamyl-Lcysteinyl-glycine, GSH) stands as the most abundant free thiol in biological systems (Hayes and McLellan, 1999). GSH has vital roles in the cell, such as response and detoxification of oxidants or other chemicals and protection of the thiol-redox balance (Xiang C et al., 2001). ASC on the other hand is a wellknown antioxidant as well that is involved in defense mechanisms against oxidative stress. Salt stress cause increased oxidation of the apoplastic ASC pool. Higher foliar ASC levels found in plants are able to tolerate oxidative stress with a better capacity (Potters et al., 2004). Analyzing the accumulation of uncharged and relatively stable reactive oxygen species (ROS), such as intracellular H2O2, is an important factor to measure membrane damage (Maruta et al., 2012).
To alleviate the damage caused by salt stress, plants generally increase the synthesis of soluble sugars and proline to be able to enhance the efficiency of osmotic regulation. Proline regulates osmotic pressure in cytoplasm and preserve the structure of the cellular components, thereby increases the ability of the plant to resist salt stress (Bates et al., 1973). Soluble sugars can increase the concentration of cell sap and increase the stress tolerance of plants (Ashraf and Foolad 2007). Changes in photosynthetic pigments are also markers of salt stress tolerance.
The chlorophyll content normally shows a downward trend after salt stress in various plants (Karimi and Yusef-Zadeh 2013). The genus Inula which is a member of Asteraceae has ethnopharmacological importance. Especially its flower extracts containing rich flavonoids, phenolic acids and sesquiterpene lactones are a potential agent to relief many ailments (stomach ache, bruises, joint pain) in many countries all over the world used in the pharma industry (Wu et al., 2015). Studies by now are mainly focused on its phytochemical and pharmacological activities of root/flower extracts however, its salt stress tolerance has not been received significant attention even though its growth are greatly affected by abiotic stresses.
Here, we have analyzed how Inula plants (falls in Magnoliopsida class) response to changes at photosynthetic pigments, various osmolytes generally in plants (proline and soluble sugars), oxidative stress (MDA and H2O2), several enzymatic and non-enzymatic antioxidants under low and high salt treatments by using above mentioned knowledge. Inula plants under higher NaCl showed a better osmotic regulation and membrane protection, which led to a stronger tolerance.

Plant Growth Conditions and Stress Treatment
Inula seeds collected from nature (Hannover, Germany) and seedlings obtained by germination were selected based on the robust morphological traits and placed in the pots containing Miracle-Gro garden mix. Right after young plants were robust enough (after 3 weeks of germination) plants were watered twice a week with Hoagland containing NaCl at 100 (low salt) and 500 mM (high salt) final concentrations, or without salt for the control (non-stressed). All experiments were conducted in an environmental chamber, with following parameters: 16 h/8 h light/dark cycle at 23 °C ± 2, 300 µmol m -2 s -1 of photon density of the leaf surfaces, and 50-80% relative humidity. Within the following 7 days, the plants were grown under salinity, and then they were harvested to conduct experiments. Leaf material was used to perform all kind of measurements and biochemical assays.

Enzyme Activity Assays
Inula leaves were powdered in liquid N2. Soluble proteins were extracted in 50mM phosphate buffer (pH 7.4), 1mM EDTA, The APX extraction buffer contained ascorbate (5mM). The homogenates were centrifuged at 12000g for 20min at 4 °C and the supernatant was collected for further enzymatic assays. APX activity was determined by rate of ascorbate oxidation at 290nm. The solution contained 50mM phosphate buffer (pH 7.4), 0.2mM H2O2 besides 0.5mM ascorbate in final reaction volume. Superoxide dismutase activity was detected with the help of the method of Dhindsa and Matowe (1981). The reaction product was spectrophotometrically measured at 560 nm. Guaicol peroxidase was monitored by the changes in absorbance at 470 nm as Urbanek et al., (1991) suggested (extinction coefficient 26.6 mM -1 cm -1 fot tetraguaiacol). Glutathione reductase activity was determined with the oxidised glutathione (GSSG) and oxidation of NADPH (Foyer and Halliwell, 1976). Total proteins were calculated by Bradford protein assay.

Non Enzymatic Antioxidants Determination
Inula leaves were harvested (500mg), powdered in liquid N2 and GSH and was measured spectrophotometrically at 412 nm as described by Griffith (1980). Ascorbate contents were determined at 265 nm with perchloric acid, NaH2PO4 (pH 5.6) and sufficient K2CO3 with 1 U ascorbate oxidase (Foyer et al., 1983).

H 2 O 2 , Proline, Sugars and Pigment Contents
Inula leaves (0.25 g) were homogenized in 5% trichloroacetic acid and 0.1 g of charcoal (activated) at 4 °C for H2O2 assay. The homogenate was centrifuged at 12,000 × g for 15 min. 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 0.75 mL of 1 M KI were used to add in 0.5-mL of the supernatant which then was measured at 390 nm (Velikova et al., 2000). Proline concentration was determined by the ninhydrin method of Bates et al. (1973) in dry leaf samples (0.1 g). Proline content was given as mg g -1 DW using a standard curve. Dried inula leaf samples were powdered in 80% ethanol. The mix was filtered and the filtrate was centrifuged at 4°. Reducing sugar content was estimated by a color change at 600 nm (Ross, 1959). Total chlorophyll (chl) and Carotenoid (car) content of inula plants have been recorded by using Arnon's equations in 80% acetone (Arnon, 1949).

Statistical Analysis
The means of indicated replicates used for data analyses for all experiments conducted in this study. Significant differences between treatments were evaluated by SigmaPlot, version 11.0 software at 5% (P ≤ 0.05) level.

MDA and H 2 O 2 Level
The concentration of MDA, a reliable biomarker of oxidative stress, was gradually increasing in parallel with the increment in salt concentration in the leaves (Figure 1). 100 and 500 mM NaCl treated leaves induced 1.6 and 2.6-fold higher than the control respectively. As with lipid peroxidation, H2O2 amount increased in both 100 and 500 mM NaCl treated gropus. H2O2 was found 2.0 and 3.2 fold higher than control plants in 100 and 500 mM NaCl treatments respectively ( Figure 1)

Figure 1
Changes of MDA, H2O2, GSH and ASC contents in Inula under different concentrations of NaCl 100 mM (low salt) and 500 mM (high salt) exposure for 7 days. WW: well-watered control, 100 mM: 100 mM NaCl and 500 mM: 500 mM NaCl. Data were shown as the means of ± SD (N = 5). Different letters indicate significant differences between experimental groups (p < 0.05). Statistical analysis was carried out using one-way ANOVA followed by Tukey post-hoc test

Antioxidant Substances
The effects of low and high salt exposure on the contents of antioxidant substances (ASC and GSH) in inula plants are shown in Figure 1. Salinity treatments caused an increase in endogenous ASC concentration. As compared to control, ASC increased by 15% and 36% in 100 and 500 mM NaCl treatments, respectively. Low salt (100 µM) treatment did not affect GSH concentration as much as high salt (500 µM) concentration. About 21% increment in GSH content was recorded in 500 mM NaCl treatment with regard to control. However, there was also no statistical difference between the two NaCl treatments (Figure 1).

Osmolytes Accumulation
Levels of two types of osmolytes commonly used by plants: proline (Pro), and total soluble sugar (TSS), was measured in the leaves of inula plants subjected to NaCl stress. The proline content was increased in 100 mM and 500 mM NaCl and recorded 2.5 and 6.1 fold as to control ( Figure 2). As compared a bc ab c Groups to control, the TSS content in leaves of inula plants was increased in 100 and 500 mM NaCl treatments. TSS was recorded 2.8 and 3.0 fold higher in 100 and 500 mM NaCl treatments. However, no significant difference found between sugar contents of 100 and 500 mM NaCl treated plants.

Figure 2
Changes of prolin and total sugar amounts in Inula under different concentrations of NaCl 100 mM (low salt) and 500 mM (high salt) exposure for 7 days. WW: well-watered control, 100 mM: 100 mM NaCl and 500 mM: 500 mM NaCl. Data were shown as the means of ± SD (N = 5). Different letters indicate significant differences between experimental groups (P < 0.05). Statistical analysis was carried out using one-way ANOVA followed by Tukey post-hoc test.

Degradation of Photosynthetic Pigments
Total chlorophyll level show significant decrease in leaves of inula plants subjected to 100 mM NaCl treatment but was disrupted by more than 50% in 500 mM NaCl treated plants (Figure 3). Both stress treatments caused significant reductions in carotenoid concentrations. Concerning carotenoid levels in the leaves of Inula plants undergoing salt stress treatments, 1.3-fold decrease was detected at 100 mM NaCl, and a 47% decrease after 500 mM NaCl treatment as compared to controls (Figure 3).

Figure 3
Changes in photosynthetic pigment contents of Inula under different concentrations of NaCl 100 mM (low salt) and 500 mM (high salt) exposure for 7 days. WW: well-watered control, 100 mM: 100 mM NaCl and 500 mM: 500 mM NaCl. Data were shown as the means of ± SD (N = 6). Different letters indicate significant differences between experimental groups (P < 0.05). Statistical analysis was carried out using one-way ANOVA followed by Tukey post-hoc test.

Antioxidant Enzyme Activity
As an expected picture generally, a major increase of all enzyme activities was obtained in response to salt stresses. SOD activity of inula plants under these different NaCl concentrations were given in Figure 4. About 51% and 67% increment in SOD activity was recorded in 100 and 500 mM NaCl treatments comparing to their control. However, there was no remarkable difference between the two NaCl dosages. GPOX activity significantly increased with increasing NaCl levels.
GPOX activity was higher in 500 mM NaCl treated plants than that of 100 mM NaCl in terms of increasing ratio. For example, the activity was increased by 100% and 246% in 100 mM and 500 mM NaCl treatments as compared to control, respectively. Regarding APX activity, it increased by 240% under high salt (500 µM) and in low salt (100 µM) concentrations by 104% as compared to its control. At last, GR activity in Inula leaf extract, gave a concentration-dependent boost in response to salinity (about 118% higher than in its respective control) measured in the presence of 500 mM NaCl.

Figure 4
Changes of antioxidant enzymes SOD, GPOX, APX and GR activities of Inula under different concentrations of NaCl 100 mM (low salt) and 500 mM (high salt) exposure for 7 days. WW: wellwatered control, 100 mM: 100 mM NaCl and 500 mM: 500 mM NaCl. Data were shown as the means of ± SD (N = 6). Different letters indicate significant differences between experimental groups (P < 0.05). Statistical analysis was carried out using one-way ANOVA followed by Tukey post-hoc test.

Discussion
It is a cornerstone in plant physiology that chlorophyll intactness in plants is directly related to plant health. Its concentration under salt stress is a sensitive indicator of the cellular metabolic state of the target plant. A decrease of chlorophyll concentration in many herbaceous and woody species under same saline environments has been reported by numerous researchers (Schiop et al., 2015;Taibi et al., 2016). A marginal reduction in chlorophyll content was detected in Inula plants subjected to two different concentrations of NaCl here in this study, was more obvious in the presence of high salt concentration. This situation appears to be a synergistic effect on the specific enzymes inhibition (i.e. Rubisco or PEP carboxylase) related to chlorophyll synthesis and the acceleration of degradation by the chlorophyllase (Kumar et al., 2017). Carotenoids, besides their role as accessory pigments which called "light-harvesting", have functions on protecting chlorophylls from photo-oxidative injury (Katarina et al., 2014). Carotenoid level was also affected in Inula by salinity meaning direct correlation between carotenoids and chlorophylls lead chlorophyll loss might be due to the carotenoids degradation. Kumar et al., (2017) detected remarkable decrements in chlorophyll and carotenoid contents in oleander plants exposed to salts, monitored more clearly in the presence of higher saline conditions. MDA is known as a reliable biochemical marker indicating oxidative stress damage. Different salt concentrations generated oxidative stress in Inula here, as supported by the increased amounts of MDA and endogenous H2O2. The increments found in the amount of H2O2 and lipid oxidation have shown that oxidative stress occurs as a secondary stress in Inula plants. Salinity leads high levels of ROS in plant cells due to malfunction of electron transport chain and accumulated photo reducing power (Hossain and Dietz 2016). This excessive electro-chemical energy can be destroyedvia the Mehler reaction, which generates ROS, such as H2O2, besides membranes damages, mirrored in elevated EL and MDA levels (AbdElgawad et al., 2016).
Cellular antioxidant system and its activation is crucial for sustaining redox balance which is a need for salt adaptation or tolerance response in plants (Nikalje et al., 2018). The activities of the target antioxidant enzymes (SOD, GPOX, APX and GR) induced in parallel with increasing salinity. SOD is known as one of the most powerful antioxidants in the system (Sales et al., 2013). We determined that the total SOD activity in the leaves of Inula was greatly increased in both NaCl concentrations. As indispensable antioxidative enzymes, GPOX and APX catalyze H2O2 to water and oxygen reactions under stress (Gong et al., 2014). Here, high dose salt treatment induced a greater increase in both GPOX and APX activities. APX and GR also support cellular redox state, by catalyzing H2O2 to water and oxygen coupled to ascorbate oxidation, and the reduction of glutathione (GSSG) to GSH by a cofactor (NADPH). In our experiments, most likely that the activation of these enzymes the plants was counteracting higher ROS levels. The results also supported a well-known notion that this enzyme system is a defense arsenal under high salinity. Our results are in conformity with findings that different concentrations of table salt induced SOD, CAT and APX activities in Nitraria tangutorum (Yang et al., 2010).
Besides appreciable contribution of the APX, GPOX and GR enzymes in counterbalancing the damages caused by ROS, supporting capabilities of metabolites (ascorbate and glutathione) is creditable as well. Considering, cellular GSH and ASC reductants involved in the defense against ROS it was not a surprise to monitor GSH and ASC increases in leaves of stressed Inula plants. Present findings form a well correlation between GSH and GR activity in Inula exposed to different salinity. Changes in GR are likely to modulate total glutathione pool. Indeed, GR activity boosted with increasing NaCl levels. The fact that NaCl stress further induces activity of GR in Inula plants, might show involvement of different metabolic routes as well as maintaining high GSH levels in the plant tissues. It is also possible that other antioxidant molecules modulate/compensate for fluctiations in the glutathione amount (Kaur and Bhatla, 2016). Our data indicated that different salt stress treatments elevated the level of ASC gradually. Higher endogenous ASC is critical for sustainable antioxidant capacity due to its role in preserving APX activity (Zhou et al., 2009). The results suggested that the increases in GSH and ASC were essential for tolerance to salt stress-induced oxidative stress.
Proline and soluble sugars are for osmotic adjustment in a cell, thus can improve growth under stresses (Singh et al., 2015). In our study, the accumulation of proline showed a linear increase under two NaCl concentrations but this increase was higher in high salt (500 µM) in leaves of inula. Similar to our data, two Mediterranean halophytes (Plantago crassifolia and Inula crithmoides) showed that high proline accumulation contributes to osmotic balance under 450-600 mM NaCl (Pardo-Domènech et al., 2016). Increased proline level under salinity in the present work, can be because of the up-regulation of proline synthesis and degredation enzymes simultaneously. Indeed, proline accumulation under stress is either because of the upregulation of proline biosynthesis gene expressions (P5CS, P5CR) or because of down-regulation of the target genes in its degradation pathway (PDH silencing) (Marco et al., 2015). Sugar accumulation is an osmotic balance pathway as well permitting plants to maintain their storage reserves (Smeekens, 2000). By detecting total soluble sugar increase in parallel with the salt stress, showed that both solutes helped buffering the redox potential of the cell and protected the cellular structures against NaCl. Inula plants may adopted some mechanisms such as synthesizing more osmolytes to rapidly adapt to that NaCl levels. The increase in osmolytes might also be related to the the ion content status of the tissue. Nikalje et al, (2018) have also proved that both proline and TSS were positively correlated with Na increase and thus possible that pathways such as protection of integrity of membranes and/or improved stability of ion transporter proteins or channels might contribute to salt tolerance as well.

Conclusion
Inula leaves exhibited well organized responses to higher NaCl related to the induction of high osmolytes (proline and TSS) and antioxidant systems to ease NaCl damage. Especially, activation of GPOX, APX and GR and non-enzymatic antioxidants (GSH and ASC) accumulation contribute to fight the injurious effects of oxidative stress as expected. Although Inula is not mainly categorized as a halophyte genus, nonhalophyte species as used in this study are nevertheless quite robust against high salt and species grown in this niche are suitable for use in pharmaceutical industries thanks to their NaCl tolerance capacity.