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Freeze-thaw Properties of β-glucan Gels

The network structure of β-glucan polymers and the presence of water have significant effects on the properties of β-glucan gels produced by freeze-thaw cycles. The properties of the gel are influenced by its fine structure, molecular size, mass fraction, number of freeze-thaw cycles, and temperature of the β-glucan. To characterize β-glucan freeze-thaw gels, we used low-field nuclear magnetic resonance (LF-NMR) to measure water proton transverse relaxation in aqueous β-glucan solutions during storage. The results indicated that microphase separation occurred during cryogelation, and three water components were identified in the cryostructure. The spinspin relaxation time was analyzed on the basis of chemical exchange and diffusion exchange theory. The location of each water component was identified in the porous microstructure of the cryogel. The pore size measured from scanning electron microscopy (SEM) images agreed with the pore size estimated from relaxation time. The formation of cryogel was confirmed by rheological. The results suggested that LF-NMR can monitor the polysaccharide cryogelation process by the evolution of spin-spin relaxation characteristics.

Introduction
Nowadays, thanks to its function to lower cholesterol [1], reduce the risk of type 2 diabetes [2,3], enhance gut health 4 , and improve immunity [5], β-glucan has been getting increasing attention, which can be extracted from cereals such as oat, barley, rye, and wheat. β-glucan is a linear polysaccharide of d-glucopyranosyl residues linked by β-(1→3) and β-(1→4) glycosidic bonds [6], whose molecular weight is reported to be between 60 and 3000 kDa 7 . Together with lichenase (EC 3.2.1.73), the oat β-glucan will release oligosaccharide fragments, which are regarded as the building blocks of an intact β-glucan chain. There exist two major oligosaccharides segments in the hydrolysate, namely, 3-O-β-cellobiosyl-d-glucose (DP3) and 3-O-β-cellotriosyl-d-glucose (DP4) [8,9,10]. The polysac charide structure, solution viscosity, and gelation behavior mainly contribute to the healthcare effects of oat β-glucan. Therefore, it's quite imperative to thoroughly understand how the β-glucan polysaccharide works in different aqueous environments. With a sufficiently high concentration, β-glucan may change from a solution form to a gel form. There are many studies indicating how aggregate structures of oat β-glucan can be in the solution and in the form of a gel, with static and dynamic light scattering [15,16], rheometry [17,18], atomic force microscopy [19], and confocal microscopy [20]. In different aqueous solutions, the effects of molecular weight, structure and concentration of oat β-glucans on its aggregation state are different. For example, the β-glucans with low molecular weight is more likely to form aggregates and gels [21,22,23,24] than the one with high molecular weight. Moreover, the gelation rate of β-glucan increases with the increase of the polysaccharide concentration in the solution. Time-domain 1H NMR has been widely applied to the study of polysaccharide solution and gel [25,26,27]. Meanwhile, more useful information on polymer dynamics and aggregation structure can be obtained through the transverse relaxation time, T 2 [28]. Adding polysaccharide to water will usually aggrandize aqueous proton transverse relaxation rate, 1/T 2 . The proton frequently exchange between water and polysaccharide hydroxyl groups, which has a significant effect on the transverse relaxation mechanism of polysaccharide aqueous solution and gel. It is possible to calculate [29,30,31] the water proton transverse relaxation time with the help of a two-site exchange model. Investigations were made to study the hydration of chitosan and the water tightly coordinated with the polysaccharide in chitosan hydrogel. Therefore, it is possible to elucidate the microstructure of native starch granule and the gelatinization of starch through the NMR relaxation and diffusion methods. In the work described in this article, different water components was identified by low-field nuclear magnetic resonance (LF-NMR) and the description of its residue's microscopic structure was made. In addition, the formation process of oat β-glucan gel was verified by rheological method, providing a theoretical basis for the practical application of β-glucan in food industry and the fine processing of oat and Highland barley, eventually making a contribution to the development of grain science. Moreover, the oat β-glucan gel can also be used as a sustained-release drug carrier. In contrast to the conventional pharmaceutical preparations, these gel carriers have many advantages, such as long treatment period, relatively low drug delivery frequency, little gastrointestinal stimulation, non-toxic side effects, few fluctuations in drugs peak period, no oral administration no drug release in the front of the stomach and small intestine, repeated drug use on gastrointestinal mucosa stimulation and reduced systemic side effects [32].

Isolation and Purification of β-glucan
Oat β-glucan was extracted from the Weiduyou 1 oat cultivar and barley β-glucan was extracted from the Highland barley cultivar using the method described by Lazaridou et al [33] with minor modification. Briefly, the bran was treated with hot 80% ethanol in water, then washed with absolute ethanol. After air-drying, the bran was used for β-glucan extraction with 52°C water, involving a thermostable amylase and a pan creatin digestion. The β-glucan was precipitated with ethanol, and the precipitate was solubilized with water and lyophilized.

Partial hydrolysis with acid
Mild acid hydrolysis was used to obtain low molecular weight polysaccharide, as described by Wood et al [12]. Two grams of β-glucan was dissolved in 200 mL of deionized water. The solution was heated to 70°C with stirring. Concentrated HCl was added to 0.1 M final. Samples were hydrolyzed at 85°C for 30, 60, 90 min, quickly cooled to room temperature, and neutralized with NaOH solution. Two volumes of 100% ethanol were added to precipitate the partially hydrolyzed oat β-glucan. The precipitates were solubilized in water, then desalted by dialysis in tubing with a molecular weight cut off 10 kDa. The dialyzed solutions were lyophilized. β-glucan samples with different molecular weights (Table 1) were used to make 4% (w/w) aqueous solutions. The solutions were prepared in the same manner as solubilization for HPSEC-MALLS analysis, except the solvent was deionized water. Two mL of each β-glucan solution was transferred into a NMR sample tube with an internal diameter of 15 mm. The tubes were sealed with PTFE plugs and stored in a refrigerator at -18°C for 21 h. Then the tubes containing β-glucan samples were moved into an incubator and allowed to thaw at 25°C for 3 h. The samples were tested after each of five freeze-thaw cycles, and then tested after every three additional cycles for 11 total freeze-thaw cycles. For rheology analysis, 5 mL of β-glucan solution was transferred into a cylindrical mold with an internal diameter of 36 mm. The solutions were treated with repeated freeze-thaw cycles and tested after 0, 1, 2, 3, 4, 5, 8 and 11 freeze-thaw cycles.

Preparation of β-glucan Frozen Gel
Various molecular weight (mass fraction is 4%) oat and barley β-glucan solutions were prepared in an 85°C water bath with stirring for 3 h. After the solutions had cooled to room temperature, they were placed in a refrigerator at 18°C, then frozen for 21 h, and thawed at room temperature for 3 h, process time for a freezing and thawing cycle, according to the request of test sample solution for the corresponding number of freezing and thawing. The prepared samples were labeled as OG0 and BG0.

Methylation Analysis
Sample processing steps: (1) β-glucan methylation with CH 3 I; (2) hydrolysis with 2 mol/L trifluoroacetic acid at 121°C for 1.5 h; (3) post-hydrolysis with sodium borohydride β -glucan reduction; (4) acetylation at 100 °C for 2.5 h; (5) quantitative determination of partially methylated sugar alcohol acetate with a 7890B/5977A GC-MS equipped with HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The initial temperature of the column was 140 °C for 2 min, then the temperature was raised to 320°C at 6°C/min for another 3 min. Partially methylated sugar alcohol acetate was characterized by GC-MS and quantified using a gas chromatograph equipped with a hydrogen flame ionization detector [34].

LF-NMR Measurements
A MiniMR NMR spectrometer (Niumag, China) operating at 23 MHz for 1H resonance was used to perform the LF-NMR experiments. All the NMR tubes containing samples were placed in a water bath at 32°C and kept for at least 15 min. Then the samples were transferred into the NMR probe with a constant temperature of 32°C. The spin-spin relaxation time, T 2 , was obtained using a Carr-Purcell-Meiboom-Gill (CPMG) sequence with a 90°-180° pulse and spacing of 0.1 ms. The 90° pulse was 18 μs, and the number of echoes was 18000. It produced a sampling space of about 4.23 s. Samples prepared under different conditions were assayed with at least three replicates. Each sample was repeatedly scanned eight times with a delay of 10 s. Signal collection and analysis were performed with Niumag NMR analysis software (Niumag, China). The relaxation signal decay curves were fitted with an exponential equation.
Where A i is the echo amplitude of the ith component at time t and T 2i is the corresponding spin-spin relaxation time. A 0 is the noise of the curve.

Principal Component Analysis of LF-NMR
Principal component analysis module in SPSS18.0 was used to analyze the LF-NMR relaxation time distribution data of 4% oat and highland barley β-glucan gel systems with different mass fractions and with different molecular weights, after obtaining the score data of principal components, the principal components with eigenvalues greater than 1 were retained, and the score graph drawn by Origin9.0 and analyzed accordingly.

Dynamic Rheometry
The rheological properties of the β-glucan solution and cryogel were investigated with a Physica MCR-301 rheometer (Anton Paar, Austria) at 25°C using parallel plate geometry (PP25/P2, 25 mm diameter, 1 mm gap). The solution or cryogel was transferred from the cylindrical mold to the lower plate of the rheometer before the test. G′ (storage modulus), G″ (loss modulus), and tan δ (G″/G′) were obtained from oscillatory measurements with 0.1% strain and frequency from 0.1 to 10 Hz. Analysis was carried out with at least three replicates.

Scanning Electron Microscopy
The β-glucan solutions and cryogel were lyophilized and a thin layer of the sample was cut carefully with a sharp blade. The microstructure of a cross section of the sample was obtained with a JSM-7500 F scanning electron microscope (JEOL, Japan). A small piece of cross section was fixed onto an aluminum stub with double-sided conductive tape and sputter-coated with gold. Then it was observed with SEM at an acceleration voltage of 3 kV. Figure 1 shows gas chromatography-mass spectrometry measurements of partially methylated glycolic acetate of β-glucan. Figure 1(a): the mole percent of OG0 (Glcp)1→ is 1.60%, the mole percent of OG0 →3(Glcp)1→ is 27.10%, and the mole percent of OG0 →4(Glcp)1→ is 71.30%. Figure 1 (b), the mole percent of →(Glcp)1→ to BG0 is 2.22%, the mole percent of (Glcp)3→ to 1 is 27.68%, and the mole percent of (Glcp)4→ to 1 is 70.10%. From these data, we calculated that the ratio of bonds (1→4) to (1→3) in oat β-glucan was 2.63, and the ratio of bonds (1→4) and (1→3) in highland barley β-glucan was 2.53.

LF-NMR Measurements
We measured spin-lattice relaxation time (longitudinal relaxation time) and spin-spin relaxation time (transverse relaxation time), both of which reflect the combination of water and matrix. The transverse relaxation time T 2 has a higher sensitivity in characterizing the fluidity of different moisture and determining the type of moisture with respect to the longitudinal relaxation time T 1 . In general, the shorter the relaxation time T 2 , the better the binding of water to the matrix, and the longer the relaxation time T 2 , the weaker the combination of water and matrix. Proton density M 2 represents the relative moisture content of different transverse relaxation times.  Table 1 in Methods) after freezing and thawing eight times. The gel system of β-glucan shows three sets of proton relaxation peaks, T 21 , T 22 and T 23 . T 21 corresponds to the water located in the physical crosslinking zone formed between the β-glucan molecules, and the transverse relaxation time is short; T 22 corresponds to the moisture located in the gel skeleton, and the transverse relaxation time is relatively long; T 23 corresponds to the water in the macropores formed by the melting of ice crystals with the longest transverse relaxation time. In the gel system, the diffusion between water molecules in different microenvironments is greatly affected by the gel network, and the diffusion exchange between the microenvironments is blocked, thus producing multiple T 2 distributions, i.e., multiple peaks ( Figure 2). The relaxation time T 23 corresponding to the peak of the largest proportion in the gel is closely related to the mass fraction. As the mass fraction increases, the corresponding relaxation time decreases, indicating a change in the moisture state and the environment. This process can be qualitatively understood as the relationship between the concentration of β-glucan and the transverse relaxation time of water. When the β-glucan concentration is high, the more active hydrogen can be exchanged in the system, and affect the transverse relaxation time of water. Moreover, because the relaxation time of the active hydrogen on the sugar chain is small, the transverse relaxation time of the water protons that undergo chemical exchange becomes smaller with the increase of the polysaccharide mass fraction. Therefore, with a short relaxation time, the gel network formed by the higher mass fraction of β-glucan in aqueous solution becomes denser, which hinders the diffusion of water molecules and reduces the lateral relaxation time [35].

Journal of Food and Nutrition Research
The 4% concentration of β-glucan in all the solutions was above the critical overlap concentration, belonging to the concentrated solution regime [36]. In concentrated solutions, β-glucan chains overlap and are entangled, thus, they are more likely to form aggregates. As shown in Figure 3, the T 21 of different molecular weight OG and BG polymers gradually rose during the freeze-thaw cycles, a condition that indicated formation of gel structure. The fast relaxation component (component 1) had a spin-spin relaxation time, T 21 , of about 5 ms for OG and 1-15 ms for BG. The inert protons on oat β-glucan were not expected to contribute to component 1 because the CPMG pulse sequence can hardly collect the rapid decay signal. Component 1 can be reasonably considered as stemming from water molecules trapped within the cross-links formed by consecutive DP3 units and stable physical entanglement points of β-glucan chains. Once the cross-links and stable physical entanglements were formed by freeze-thaw cycles, they became stable structures and could not be altered easily by further freeze-thaw treatment. Therefore, T 21 remained essentially constant during the cryogelation process, even though the values have some fluctuation. The spin-spin relaxation of this group of water protons is mainly modified by chemical exchange with labile protons of polysaccharide in the cross-links and stable entanglement points. The relatively small value of T 21 reflects the low mobility of oat β-glucan chains in the network skeleton. To some extent, these water molecules can be regarded as an integral part of the network skeleton structure. Component 2 of different molecular weight β-glucan changed as follows: The spin-spin relaxation time, T 22 , distributed from 35 to 160 ms for OG and 10 to 120 ms for BG, which means higher mobility of this group of water protons than for component 1. Thus, component 2 is considered as water confined in the interstitial space between β-glucan chains. β-glucan aqueous solutions experience a slow freezing process at -18°C. During freezing, ice crystals gradually grow larger and the concentration of β-glucan increases in the liquid phase, thereby facilitating interaction between β-glucan molecules. In the subsequent thawing process, the rise in temperature enhances the dynamics of β-glucan interactions; meanwhile, the temperature is still below the freezing point for a period of time, and the ice crystals remain in a solid state, keeping a high concentration of β-glucan in the liquid phase. The freeze-thaw cycles result in the formation of water pools surrounded by thin walls of concentrated cryogel, similar to a cell structure. Water molecules trapped in the interstitial space between aggregates of oat β-glucan chains are assigned to component 2. The spin-spin relaxation time, T 22 , first increases and then shows a declining trend with increasing freeze-thaw cycles. The increase of T 22 may be an indication of more open association of β-glucan aggregates, and the decrease of T 22 is probably caused by a denser gel phase compressed by ice crystals. At the least, the change of T 22 indicates the porosity and heterogeneity during the cryogelation. There was no clear relationship between the molecular weight of β-glucan and the corresponding T 22 . But β-glucan of lower molecular weight was more likely to form a gel. Figure 3(e) shows the variation of spin-spin relaxation time of component 3, T 23 , which reflects the water in the pores formed by the melting of ice crystals in the gel. The initial values of T 23 approached the values in fresh β-glucan solutions, thus, component 3 was considered as bulk water in the aqueous systems. The T 23 of each molecular weight at different stages changed as follows: Freeze-thaw treatment caused a rapid increase of T 23 at the first stage, which meant fewer labile protons on the polysaccharide were available to exchange with bulk water protons. Fewer labile protons cause an increase in T 23 because T 23 is mainly determined by chemical exchange. Higher mobility produces more smaller-sized β-glucan entering the gel microphase and causes a further rise in T 23 . T 23 begins to present a significant rise after about four freeze-thaw cycles, and the rise is more significant for lower molecular weight samples.
With more freeze-thaw treatments, the β-glucan concentration in bulk water gradually increases. Except for OG0, freeze-thaw treatment caused a slight increase in T 23 of OG30, OG60, OG90 at the first stage. The T 23 of OG0 kept rising when it was frozen and thawed two to four times. In the process of freezing and thawing 2-4 times, T 23 basically reached equilibrium; after four freeze-thaw cycles, the T 23 of various glucans was in balance. The smaller the molecular weight, the smaller the T 23 of OG, and the smaller the molecular weight, the larger the T 23 of BG. This difference is caused mainly by the differences in molecular weight and molecular structures of the two polysaccharides. The initial values of T 23 approached the values for fresh oat β-glucan solutions, so component 3 was considered as bulk water in the aqueous systems. It is surprising that T 23 is lower in the aqueous system containing small-sized β-glucan at the beginning of freeze-thaw treatment. Although the lower molecular weight β-glucan aqueous system appeared less viscous, the mobility of water therein seemed to be lower than the water mobility in the more viscous solution having larger-sized oat β-glucan. A likely reason for this abnormal result lies in the self-aggregation of oat β-glucan; β-glucan of smaller size is more prone to aggregate than larger polysaccharide molecules. The aggregation may result in a suspension or a network, which causes a decrease in the mobility of oat β-glucan molecules and the slight lowering of the T 23 of water interacting with these polysaccharide aggregates after the first several freeze-thaw cycles. The leveling of T 23 (Figure 3e) and T 21 (Figure 3a) indicates that the cryostructure has changed little after 10-13 freeze-thaw cycles.
As shown in Figure 4a and Figure 4b, the spin density, A 21 , of component 1 gradually increased with increasing number of freeze-thaw cycles, a result that reflected an increase in cross-links and stable physical entanglements. Small-sized BG molecules form a network skeleton earlier compared with their larger counterparts. Smaller BG seemed to form more network skeleton during cryogelation. Clearly, after 11 freeze-thaw cycles, BG0 produced a network skeleton significantly smaller than the other small-sized samples. It is generally considered that barley β-glucan with a small size has higher mobility of chains, facilitating the formation of cryogel structure. The formation and increase of cryostructure deduced from the presence of component 1 and increase in A 21 agree with the results obtained with traditional methods. As shown in Figure 4c and Figure 4d, spin density, A 22 , increased with the number of freeze-thaw cycles, reflecting the increase in the cryogel microphase. A 22 showed an inverse relationship with the molecular weight of oat β-glucan at all cycles; demonstrates that oat β-glucan of small size produces more cryogel microphase compared with larger polysaccharides. This result has been verified by others. A 22 increased sharply and then leveled, indicating that the quantity of cryogel microphase approaches a maximum and remains constant after eight freeze-thaw cycles. A 21 also showed the same trend, and eight could be considered as a critical number of freezethaw cycles.
After 11 freeze-thaw cycles, the spin density, A 23 , of the bulk water decreased from nearly 98% to about 80% for OG and from 100% to about 75% for BG in β-glucan samples with decreasing molecular weight. The reduction of bulk water ratio was due to the increase of cryogel microphase and increasing amount of water entrapped in the gel microphase. That is, the bulk water gradually converted into entrapped water during cryogelation. Figure 5 shows the scores of OG(a) and BG(b) with different molecular weights after eight freeze-thaw cycles. Each circle represents the overall characteristics of a particular molecular weight gel. The differences in gel samples of different molecular weights can be scored by principal component analysis. The spacing distances in Figure 5 reflect the fact that the greater the difference in molecular weight β-glucan gel, the greater the difference in gel properties. Principal component analysis can better distinguish gel samples of different molecular weights. For OG and BG, the β-glucan gel samples that were hydrolyzed for 0 min and 30 min were concentrated on the left side of the PC1 axis; the β-glucan gel hydrolyzed for 60 min and 90 min was concentrated on the right side of the PC1 axis, indicating that there was a critical molecular weight between the hydrolyzed β-glucan samples for 30 min and 60 min. Although there was no significant difference on the PC2 axis, there was a significant difference in the PC1 axis, indicating that molecular weight has a significant effect on the formation of β-glucan gel [37].

Analysis of Rheological Properties
The effect of an applied stress on a fluid can be expressed in two ways, namely elasticity and viscosity, which denote G' and G", respectively. G' is the storage modulus or elastic modulus, which reflects the ability of a polymer to deform with the change in external force. G" is the loss modulus or the viscous modulus (Pa) which reflects the energy loss caused by the internal or inter-molecular stretching of a polymer when the external force changes. Tan δ = G"/G' (δ is the loss angle) is an index of the viscoelastic properties of the system. Generally, tan δ = 1 is the limit. The larger the tan δ, the more dominant the viscous component, showing more liquid solid properties [38]. Figure 6 shows the viscoelastic properties of gels formed by different molecular weight OG(a) and BG(b) at 4% concentration and after eight freeze-thaw 8 cycles. The values of G' and G" have correlate with the molecular weights of the β-glucan gels; the smaller the molecular weight, the larger the values of G' and G". Although G' of the BG0 gel increased with the increase in oscillation frequency, the G' and G" of the other molecular weight β-glucan gels did not change with increased oscillation frequency. This difference may be because the molecular weight of BG0 is larger. The molecular weight of the hydrolyzed BG was small, and its gel form has better gel properties compared with unhydrolyzed BG0. Thus, the viscosity behavior of the fluid decreases and the characteristics of elastic behavior enhances with a decrease in the molecular weight of β-glucan and further confirmed that β-glucan having a smaller molecular weight is more likely to form a gel. Figure 6. Rheology of 4% OG (a) and BG (b) gels with different molecular weight β-glucans after eight freeze-thaw cycles.

Scanning Electron Microscopy
In β-glucan aqueous solutions, water forms a large number of hydrogen bonds with the hydroxyl groups of the polysaccharide, and the hydrated β-glucan macromolecule moves in an aqueous solution and forms a circular, helical, or double helix structure by folding and crimping. Then, by stretching, a certain part of the molecules is arranged in a straight line to form a sugar chain and a large number of β-glucan molecules are bonded at different points to form a three-dimensional network filled with water molecules to form a gel. Figure 7 shows SEMs of different molecular weight oat β-glucan and barley β-glucan gels. Clearly, β-glucan gel is a type of three-dimensional network, and the porous structure formed has a certain regularity related to molecular weight. Samples with smaller molecular weights have higher mobility and diffusivity, and it is easier to form a three-dimensional network. In addition, the small molecule β-glucan chains contain a small proportion of inactive fragments, and the degree of intramolecular action is low, hence the probability of effective collision between chains increases, and the chances of collision and entanglement between molecules increase.

Conclusions
In this study, the imperative effects of the network structure of β-glucan polymers and the presence of water has been found on the properties of β-glucan gels after specific freeze-thaw cycles. What has also been found is that three groups of relaxation components with transverse relaxation times T 21 , T 22 and T 23 ; the corresponding signal peak proportions were A 21 , A 22 and A 23 . With the increase of freezing and thawing cycles, T 21 , T 22 and T 23 of different β-glucan mass fractions and molecular weights generally increased at first and then did not change noticeably. However, T 22 and A 22 of Highland barley βglucan increased after either three or five cycles of freezing and thawing. A 21 and A 22 of different β-glucan mass fractions first increased and then did not change noticeably, and A 23 first decreased and then did not change noticeably. The variation of gels with different molecular weight β-glucan and different numbers of freeze-thaw cycles was distinguished by principal component analysis of the relaxation distribution curve. Rheological studies showed that the larger the mass fraction and the less the molecular weight, and that the larger the elastic modulus were and the more viscosity modulus it shows, indicating that more cross-linked structures were formed in the gel. The result of scanning electron microscopy indicated that the gel structure was being denser with the molecular weight becoming smaller, and the gel network was being looser with the molecular weight becoming larger.


extfieldsofstudy: Physics Chemistry

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

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Seaweed extract (Sargassum polycystum) as a preservative on sunscreen cream with the addition of seaweed porridge

The use of sunscreen is highly recommended to protect the health of the skin from ultraviolet radiation. Most commercial sunscreens contain artificial preservatives such as methylparaben, which can be detrimental to health. Preservatives in sunscreen can be substituted with natural ingredients, in which Sargassum polycystum ethanol extract is offered as an alternative in this research. The preservative is made from S. polycystum extract with addition of seaweed porridge of 1:1 S. polycystum and Eecheuma cottoni mixture. The quality of cream is tested using total microbial test, durable power prediction, antioxidant and SPF value. Its physical stability is tested through sensory testing, pH measurement, cyling test and centrifugal test. Butylated hydroxytoluene (BHT) is used as control and is then treated with methylparaben and the extract for comparison, the extract was found to have comparable microbial results with 8 weeks of preservation compared to methylparaben (9 weeks). In addition, the cream shows stability up to one year, a balanced pH according to SNI, shows no foul odor, antioxidant activity (IC50 ) of 105.42, SPF value of 2.00, and moderate to favorable consumer acceptance.

Introduction
Seaweed that grows in Indonesian waters is recorded around 555 species. One of the potential seaweed as a cosmetic raw material and its abundant availability is Eucheuma cottonii and Sargassum sp. Food and Aquaculture Organization (FAO), reported that Indonesia was the second country after China in seaweed cultivation production in 2013 which amounted to 34% of the 26,896,004 tons produced by the world [1]. Public awareness of the importance of skin health care is a driving factor for increasing demand for skin care cosmetic products. The use of skin care cosmetic products is one of the efforts to protect the skin from the negative effects of weather conditions. Air humidity in Indonesia can reach 80% with temperature [2]. Continuous sun radiation will cause black spots to appear. Sunscreen cream products have been widely developed and produced on the market, but there are some concerns including the use of hazardous chemicals that do not meet the specifications of the products produced with those on the label. Cosmetics generally contain a mixture of chemical compounds and not many come from natural sources [3]. The demand for cosmetics from herbal ingredients is currently growing very rapidly [4]. This expansion is due to the availability of raw materials from nature. Raw materials for aquatic products that have the opportunity to be developed into cosmetic products are seaweed. Sargassum sp. have greater antioxidant activity compared to Caulerpa racemosa, Ulva lactuca and Gracilaria types in the case of IC50 with 1.08±0.83, 15.05±0.61, 103.73±0.59, 24.22±0.87 μg/mL respectively [5]. Sargassum sp. is a type of brown algae  [6]. Sargassum sp. contains fucoidan and phenolic components that are able to capture free radicals [7].
Red seaweed E. cottonii contains phycocyanin compounds which contain mycosporine acid (MAAs) and consists of imine derivatives containing aminocycloheximine UV absorbent chromophore [8]. Red seaweed contains antioxidant compounds that can inhibit the penetration of strong UV light into tissues or cells. In addition, E. cottonii antioxidant activity showed IC50 of 105.04 μg/mL [9]. The active components produced include flavonoids, phenol hydroquinones and triterpenoids which are thought to be potential compounds used as raw materials for sunscreen creams.

Sampling
E. cottonii used for porridge in a dry form and at a maximum harvest age of 45 days. S. polycystum seaweed was taken in the waters of Banten Bay, Serang Regency, Banten Province. S. polycystum seaweed was cleaned and sorted from sand or objects that are carried during the picking process and washed with sea water. Seaweed that has been washed with sea water, then rinsed with fresh water that flows to remove the salt or sand content that is attached. The seaweed was then stored in a styrofoam container in the transport process to the laboratory. S. polycystum was extracted by single maceration method using ethanol to obtain its active compound and its yield was determined.

Antibacterial activity test
Antibacterial activity testing included the stages of preparation of test bacterial rejuvenation, identification of test bacteria and testing stages. The stages of test bacterial rejuvenation preparation were a modification of a standard reference from NCCLS or the National Clinical Laboratory Standards Committee which included the manufacture of PCA solid media, the manufacture of NB liquid media and using Staphylococcus aureus bacteria and Escherichia coli as standard test bacteria [10].

Preparation of Sargassum and E. cottonii seaweed porridge
Preparation referred to the previous research [11]. The process of making S. polycystum porridge. Carried out through three stages: washing, soaking and draining. S. polycystum seaweed. Those that had been washed and dried were rinsed then soaked for 12 hours using distilled water. S. polycystum, the 12 hours soaked then drained. S. polycystum porridge, done by mixing S. polycystum, and distilled water, then homogenized using a blender.
E. cottonii seaweed preparation was done through four stages, namely washing, bleaching, soaking and draining. E. cottonii washing was done with running water to get clean seaweed from foreign objects such as sand, wood, twigs and sticking dirt. E. cottonii bleaching was done to get a white and attractive appearance. Blanching using a combination of distilled water and lime (CaO) 0.5% for 30 minutes while continuing to knead to help speed up the bleaching process. E. cottonii was rinsed again and continued the process of soaking E. cottonii with distilled water for 12 hours. The next process was making porridge by mixing E. cottonii and aquades, then homogenizing it using a blender.

Making sunscreen cream preparations
The ingredients used in making cream preparations were divided into two phases. Oil-soluble ingredients include emulate, liquid paraffin, stearic acid, and BHT dissolved until homogeneous at a temperature of ±75°C called oil phase (preparation 1). Simultaneously, water-soluble ingredients include glycerin, TEA, distilled water, and 1:1 S. polycystum and E. cottoni seaweed slurry preparations dissolved until homogeneous at ±75ºC called water phase (preparation 2). After that (preparation 1) and (preparation 2) homogenized and reached the same temperature ±70ºC, mixing was done until it was formed (preparation 3) in the form of a homogeneous cream [12].
In this study modification was carried out by replacing methyl paraben with extract of S. polycystum as preservative in the cream of sunscreen seaweed. Then do (preparation 3) with 3 treatments. The first treatment was given the addition of methyl paraben and BHT, the second treatment was given an addition of S. polycystum and BHT extracts, and the third treatment was only added with BHT (control) and then homogenized for ±10 minutes at 40ºC. Fragrance was put into 3 treatments of the cream preparation and homogeneous for ±3 minutes.

Sampling
Sampling was done by diving directly at the sea in the depth of 30-100 cm from the coastline. S. polycystum was then cleaned and sorted from sand or objects that are carried during the picking process and washed with sea water. Seaweed that has been washed with sea water then dried and stored in a styrofoam container and the extraction method for S. polycystum carried out in this study is single maceration using ethanol.
In the maceration process the crude extract of ethanol from the evaporation results in a characteristic paste-like solvent with a distinctive aroma. In color characteristics, the relative color was brownish, the yield was 2.73% in the first replication and 3.96% in the second replication. The percentage of yield produced from macroalgae extraction using ethanol solvents ranged from 2-3%. Based on these data it can be identified that S. polycystum contains active compounds which are relatively soluble in polar solvents.

Preparation of E. cottonii and S. polycystum
Porridge Preparations S. polycystum slurry preparation through washing, soaking and draining processes, but not through bleaching. The taking of S. polycystum was done directly by diving, so that S. polycystum was used fresh. Fresh seaweed has a higher antioxidant activity compared to dry seaweed [13].
The immersion process of E. cottonii and S. polycystum was carried out for 12 hours to remove residual lime (CaO) in the bleaching process of E. cottonii. Another goal of immersion is to produce gel on seaweed and ensure the seaweed is clean before making a slurry preparation. The slurry preparation was made by homogenizing between seaweed and distilled water with a ratio of (1:1) using a blander.

Durable power test
The total microbial testing results showed that microbial colonies found in cream preparations from week 0 to week 4 were still below the total microbial limit so that the cream could be used. The total microbial limit in the cream required by SNI 16-4399-1996 is a maximum of 1.0x102 colonies/gram.  Based on the linear graph ( figure 2-4), the results of the TPC test can be predicted that at 7 weeks cream with BHT treatment was rejected, which means that it does not meet the standard, while the extract treatment + BHT was rejected at week 8 and methyl paraben + BHT was rejected at 9 weeks. That the use of methyl paraben preservatives and S. polycystum extracts could inhibit microbial activity in cream compared to non-preservative cream.

Antioxidant activity
Antioxidant activity of seaweed sunscreen cream + BHT treatment was able to reduce free radicals with a concentration of 50%, with IC50 values of 105.42. The addition of slurry E. cottonii and S. polycystum, as well as S. polycystum extracts were thought to have an influence on the IC50 value of cream preparations. Seaweed has a phenolic component and contains antioxidants that can fight free radicals by donating one or more electrons to free radicals [14]. Free radicals are atoms or molecules that are very unstable and reactive and damage the tissue [15].
The smaller IC50 value shows the greater antioxidant activity in the test material. A compound is said to be a very strong antioxidant if IC50 values are less than 50 ppm, strong for IC50 between 50-100 ppm, while if IC50 is worth 100-150 ppm and weak if IC50 is worth 150-200 ppm.

SPF sunscreen cream value
The value of Sun Protection Factor (SPF) shows the effectiveness of a sunscreen cream in protecting the skin. The results of the calculation of the SPF value of sunscreen cream preparation are presented in table 1. The classification of the sunscreen ability level is minimum if the SPF is between 2-4, 4-6 moderate, 6-8 extra, 8-15 maximum, and ultra if the SPF is more than 15 [16]. The extract has a SPF value of 2.00 which is still in the range of moderate sunscreen protection, thus it is potential to use in sunscreens.

Sensory test
Sensory testing is a subjective test that is implemented with the value of consumer preference for product acceptance. Sensory testing used a hedonic scale with untrained panelists aged 20-35 years totaling 30 people. The results of sensory test can be seen in figure 5. From this figure, we can conclude there is no significant difference between cream with extract and cream with methylparaben in sensory testing. It is safe to assume that the extract is potential to replace methylparaben as an additive.

Appearance
The sensory results showed that the panelists' response to the appearance of sunscreen cream gave different ratings. It can be concluded that the cream + BHT extract sample obtained the lowest value of 4.8, because the cream extract + BHT had a less attractive appearance, while the panelists' favorite value on the appearance of the cream sample methyl paraben + BHT and BHT ranged from 6.5 to 6.6 which means that the panelists give an assessment between normal and somewhat like.

Color
Color is one of the parameters of visual observation attached to a product. Color can be one of the assessment factors in the selection of a product by consumers. Panelists' preference for the cream color of methyl paraben + BHT and BHT samples ranged from 6.3 to 6.5, which means the panelists gave a rather favorable assessment. Whereas the + BHT extract sample had the lowest preference value because the addition of Sargassum polycystum extract caused the cream color to dark green.

Flavor
A pleasant and easily recognizable aroma will generally be preferred compared to an unrecognized aroma. Fragrance or fragrance can duplicate the desired aroma [17]. The higher the percentage of aromatic compounds, the longer the intensity and aroma are obtained. Panelists' favorite score on the aroma of the cream ranged from 5.1 -6.4 which means that the panelists gave an assessment between rather like to like.

Homogeneity
Homogeneity is a parameter to see the effectiveness of evenly or not mixing ingredients in the product. The panelists' preference value for the cream homogeneity of methyl paraben + BHT and BHT samples was 6.5, which means the panelists gave a rather favorable assessment, while the cream + BHT cream sample had the lowest value of 6 because the addition of extract made extract + cream BHT is more dense than methyl paraben + BHT and BHT cream.

The pH of sunscreen cream preparations
The results of pH measurements for 10 weeks at different temperatures are at low temperatures (4±2°C), room temperature (28±2°C), and high temperatures (40±2°C). The pH value of the cream addition of seaweed porridge with three treatments namely methyl paraben + BHT cream, extract + BHT and BHT has increased with the length of storage time, but still in the range of skin pH balance value and in accordance with SNI Number 16-4399-1996. product for the possibility of crystallization and as an emulsion test on cream as an indicator of emulsion stability [18]. The results of the cycling test showed that there was no phase separation in the cream emulsion, discoloration and no change in aroma the oil phase and the water phase in the cream preparation so that the cream can be mixed homogeneously and remain stable at extra temperature changes, the color changes that occur in the cream preparation are due to the oxidation process of vitamin C. The oxidation process will occur faster if the cream is exposed by air, light and hot. After experiencing the color oxidation process the cream can turn into a bit darker.

Centrifugal test
Centrifugation was carried out at a speed of 3,800 rpm for 5 hours. The results of observation after centrifugation testing showed that seaweed cream preparations with methyl paraben + BHT treatment, extract + BHT and BHT. No color and odor changes or phase separation. The absence of phase separation in cream preparations is due to the surfactant used to protect oil droplets on the cream preparation so that the oil phase and water phase are well mixed [16].

Conclusion
Seaweed (S. polycystum) extract with the addition of seaweed porridge has potential to replace methylparaben as an adequate natural preservative in sunscreen cream.


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The modeling system of horticultural commodity development in Pinggan village, Bangli regency, Bali Province

Pinggan Village, located in the province of Bali, is located in the highlands, this village is a producer of horticultural commodities in the region of Bali. in discussing horticultural issues, it is also important to know the typical characteristics of Horticulture products, namely: easily damaged in transportation, overflowing / seasonal, rarely in other seasons, and sharp price fluctuations. Modeling system in the development of horticultural commodities in the province of Bali has not been done much. This research is oriented towards achieving goals and ease of operation, for this reason, the analytical method used will be adjusted according to the type and purpose of the research itself. The descriptive analysis method used in this type of research has the objective of obtaining information and factual descriptions and phenomena systematically and accurately, such as regional profile assessments, social engineering, and market potential assessments. The results of this study indicate that some constraints on the production of horticulture crops are that cultivation still needs to be improved in three commodities, including the development of fertilization, maintenance of a healthy garden environment, prevention of pests, water problems and markets.

Introduction
One indicator of food security is access to food. For non-food producing regions, food security can be achieved through increased access to food. National food needs continue to increase along with the growth of the Indonesian population. This pressure is getting bigger, because national food is only based on rice, with the need of 32.49 million tons (2010). Thus, the development of local staple food and its industrialization is indispensable for national food security. Currently, the planting area of Indonesian vegetables is only 40 m2 per capita. This figure is far behind from China which is 200 m2 per capita and Thailand which is 100 m2 per capita. Farmers' ownership of cultivated land is also low.
Indonesian farmers on average have only 0.2-0.3 ha per farmer. In addition, the consumption of vegetables in Indonesia is only 40 kg/capita/ year. This figure is lower than the standard World Food Agency (FAO) which pegs 80 kg /capita/year. As for import, in 2011, the import value of horticultural products reached 17.6 trillion. For 2014, it is estimated that the volume of horticultural imports will reach 600 thousand tons, up 50 percent compared to last year's 400 thousand tons [1]. The role of horticulture is: a) Improving the nutrition of the people, b) increasing the country's foreign exchange, c) expanding employment opportunities, d) increasing farmer's income, and e) meeting the needs of beauty and environmental sustainability. However, in discussing horticulture issues, it is also important to note the typical characteristics of horticultural products, namely: a). (B) voluminous, c) perishable in transportation, d) overflow / seasonally and rarely in other seasons, and e) sharp price fluctuations [2]. By knowing the benefits and characteristics of the typical, in the development of horticulture in order to succeed well then needed a deeper knowledge of the horticultural problem. Village Pinggan Kintamani District, Bangli Regency Bali Province is located in the highlands, not the main food producer but is the horticultural center in Bali area. So far, the horticultural product market in the region has been competing with horticultural supplies from Java. Some phenomenon of horticultural agribusiness problem in research location is: not yet optimal technique of cultivation, often The occurrence of excess supply (excess supply), not yet familiar with processing technology, not optimal the role of institutions at the level of farmers and not yet the formation of networks with stakeholders. Based on that, it is very suitable if, in the Pinggan area, the clusters of agro-industrial products of horticulture derivatives (use of fruit, leaves, stems, and plants) are developed. Therefore, it is necessary to conduct comprehensive activities which include: (1) developing technology of horticultural cultivation and development of horticultural foodstuff (RPL), (2) social engineering and technology of horticultural derivative products to increase added value; (3) dissemination of technology and social engineering Implementation of RPL, (3) commercialization of horticultural derivative products and developing marketing systems; and (4) institutional strengthening at the farm level through networking arrangements between farmer organizations (farmers and cooperatives), governments and stakeholders. The four things are done simultaneously starting from the provision of raw materials, social engineering, and technology, market development by involving various stakeholders. It is expected that at the end of this research activity can bring clusters of agroindustry and formed Area of Sustainable Food House (KRPL) based on horticulture. The success of this program will trigger the growth of rural agroindustry made from raw and local technology, so that it can increase the absorption of manpower, increasing the income of the community which ultimately improve the access of better food. The high level of food access will strengthen food security in the community in Pinggan Village, Kintamani District, Bangli Regency, Bali Province. In addition, derivative products produced based on Zero Waste so as not to cause waste but all components of horticultural commodities can be utilized.
Long-term research objectives are expected to have a significant impact on income generation, resulting in increased access of farm households to family food. Although physically staple food products are not produced in Pinggan, but the availability of pagan is sufficient through access to affordable food from family income. Besides, the increasing of agro-industry activity and sustainable food house will have an impact on economic growth and job opening so that it will increase food security. In particular, this activity aims to develop horticulture based agroindustry in the Village area Pinggan Kintamani District with a variety of activities of horticultural crops namely chili, tomato, and pumpkin Some of the phenomena of horticultural agribusiness problems in research locations include: not optimal farming techniques, frequent excess supply, not yet familiar with processing technology, the role of institutions at the farm level is not optimal and networks with stakeholders have not been established.
Based on that, agro-industry clusters of horticulture derivative products are developed in the suburbs (utilization of fruit, leaves, stems, and plants). For this reason, comprehensive activities are needed that include: (1) developing horticulture-based sustainable food cultivation (RPL) technology and development, (2) social engineering and horticultural derivative technology to increase added value, (3) conducting technology dissemination and social engineering the application of RPL, (3) commercialization of horticulture derivatives and developing a marketing system, and (4) institutional strengthening at the farm level through the establishment of networks between farmer institutions (farmer groups and cooperatives), government and stakeholders. The four things are done simultaneously starting from the provision of raw materials, social engineering, and technology, market development by involving various stakeholders.

Implementing research data
In the process of implementing this research, data sources are obtained through primary data and secondary data relating to the implementation of activities in the modeling system. Primary data is collected from sample farmers, administrators or managers from several market players in horticulture plants and secondary data obtained from the statistics center, processed data from the Department of Agriculture and horticulture of Bali Province, and reference literature. The data collection techniques were carried out by 1) questionnaire spread, 2) interviews and 3) forming Focus discussion groups (FGD). The use of questionnaire techniques in this study has several objectives, namely to obtain information relating to the purpose of research and to obtain information that has high reliability. In conducting interviews, the technique used is to ask directly to the sample/respondent and aim: To create a good relationship with respondents / samples, to be able to convey all questions correctly and correctly and apply direct questions, to be able to record all verbal responses from respondents / samples carefully and clearly by asking again if it is not clear, and to explore additional information related to the purpose of this study.
FGDs are needed to obtain more comprehensive information or data because it involves several people in discussions that invite active participation from discussion participants. Discussion participants came from the farmer level, farmer organizations (subak), company-level managers or other companies. The study team acted as the facilitator in this discussion.
The library study conducted in this study aims to obtain information/data relating to the purpose of this study, namely through the study of research books or scientific magazines and others that have been published or documented in the library.
The purpose of data collection:  Review, observe, measure and record parameters relating to the application of fair trade methods in the healthy rice trading system;  Determine the environmental quality of various environmental parameters that are expected to be significantly affected by project activities,  Assessing, observing, measuring, and recording components of planned activities that are expected to cause a large and significant impact on the surrounding environment,  Predict changes in initial environmental quality due to project activities. The location of data collection is determined in the designated area spread in the village of Pingan. Some of the data collected involve related technical, social, agro-economic and business variables.
The social indicators examined in this study are on social ties between farmers, social ties between farmers and farmer organizations (subak), social ties between farmer managers, and social ties between farmer organizations and the government. Agro-economic indicators are carried out on wetland areas, planting intensity, planting patterns and planting schedules, choice of varieties, use of agricultural inputs, use of agricultural tools and machinery, use of labor, use of cultivation technology, input prices, processing, products, storage, marketing, and output/yield prices. On business indicators, access is made to users to the horticulture market, and to access finance.

Data analysis
Data analysis will be carried out by agricultural analysis and Value Chain Analysis (VCA) in addition to descriptively. Value Chain Analysis is one of the attachment concepts to increase activity and maximize product value in the order of the supply chain. The stages of VCA activities are as follows:  Presenting a real problem situation,  Analyzing the situation of stakeholders together with analysis of value chains, institutions, social systems,  Arrange the sequence of problems faced,  Perform repairs and solutions to existing problems through system identification, model design, suggestions for improvement Table 1. Results of research on rural horticulture problems.

No
Problem Alternative Activity 1 The lack of availability of organic fertilizer Application of SOP for making organic fertilizers among farmers/farmer groups

Research approach
With a research approach that is more oriented towards achieving goals and ease of operation, the analytical methods used are tailored to the type and purpose of the study. The descriptive analysis method is used for this type of research which aims to obtain information and description of the factual conditions and phenomena of events systematically and accurately, such as regional profile assessments, social engineering, and market potential assessments. The results of the analysis are presented in the form of numerical numbers, tables or graphs so that they are easier to understand, meaningful, and able to provide useful information [3]. Spatial information and attributes from the results of mapping the production potential and market for horticultural commodity industries are visualized using Geographic Information Systems (GIS). According to Turban GIS is a computer-based system for capturing, storing, checking, integrating, manipulating and displaying data with digital maps [4]. The GIS function is to improve the ability to analyze spatial information in an integrated manner for planning and decision making. GIS can provide information to decision makers to analyze and apply spatial databases so that it is easier to see phenomena with a better perspective [5]. For the development of cultivation of horticulture crops, harvesting tools and their products use the comparison method. Comparative research aims to find answers fundamentally about causation, by analyzing the causal factors or the emergence of certain phenomena. With this method generalization of the level of comparison can be made based on a perspective or framework only so that it can determine which alternative is better and preferred to be chosen [6]. The ISM (Interpretative Structural Modeling (ISM) method is used to determine the appropriate institutional structure and form in horticultural management. The ISM technique is a group learning process in which structural models are produced to photograph complicated problems through carefully designed patterns using patterns designed using graphics and sentences This method is done by arranging hierarchies and classifying sub-elements. The basic principle is to identify from the structure  6 of institutional systems that provide high-value, effective and operational benefits [7,8]. This horticultural management system model is developed using a system approach method. developed to build system configurations and scientific relationships Heuristic techniques are suitable for handling unstructured and difficult to solve problems, although this technique does not have a search algorithm for op that is definitely a lead solution but has rules that can explore the most promising search space, namely space for optimal or near-optimal solutions that can be used to solve complex problems. In heuristic algorithms, approaches are easier, faster and have good approximate results developed [7]. The resulting model is then poured in the form of computer applications that are integrated with the operating system on mobile phones. Many socialization and community empowerment activities during the research, such as the development of added value in the bamboo industry, management development and workshops of PLTB were used in participatory methods through internal training. This method is very effective because of the direct involvement of the community (community) in activities. The effectiveness of this activity was then assessed using rapid assessment techniques developed using assessment techniques [9].
The problem discussed here requires the use of the right research approach. A system approach is a comprehensive approach that can handle complex and uncertain systems. According to the system approach, there are six stages of analysis before synthesis, namely (a) needs analysis; (B) system identification; (C) problem formulation; (D) system modeling; (E) model verification and validation; And (f) the implementation model.
System identification is a chain of relationships between system statement requirements and specific statements of problems being resolved. Identification systems are studied in the form of diagrams. The diagram used is then translated into an input-output diagram (black box diagram). The causes and effects of various related systems and their interactions are those that explain system requirements and problems. Administratively, this village is divided into one work area of the Banjar Service, namely the Banjar Pinggan Service. In running the government as an assistant at the Banjar Village Service level. In addition, it consists of villages suitable for village planning.
Economically, the suburban village can be mapped to 500 ha of horticulture areas, 350 ha of forest, 410 ha of plantations, ravines and critical areas of protection for 200 ha, and the rest for public facilities and settlements.
At present, the 700 ha of village land in the agricultural area is planted with 300 hectares with 646 farmers. Squash ownership ranges from 25 to 50 hectares/farmer. Planted with Chile covering an area of 30 ha from 646 farmers. Farmers' land for Chilean cultivation ranged from 50 to 75 hectares/farmer and planted TOMAT with an area of 50 ha from 646 farmers. Farmers' land ownership for Chile ranges from 15-25 acres/farmer. But there are still many obstacles that need to be corrected.
Constraints on the production of horticultural crops are still important, including fertilization, maintaining a healthy garden environment, water problems, and markets.

Conclusions
Administratively Pinggan village is divided into one working area of Banjar Dinas namely Banjar Pinggan Service. In running the government as a maid at the level of Banjar Pinggan Village Service. In addition, it consists of three villages Preparation of other villages which according to the prevailing provisions is clearly not justified so often lead to conflict between the village Preparation with Pinggan Village residents.
Economically, Pinggan Village area can be mapped to 500 ha of horticulture area, 350 Ha forest, 410 ha plantation, chasm, and critical area protection 200 Ha, and the rest for public facilities and settlement.
Currently, the area of agricultural land in Pinggan Village 700 ha is planted with 300 hectares of 646 farmers. Ownership of farmers' land for planting squash ranges from 25 to 50 acres/farmers. Planted with chili area of 30 ha from 646 farmers. Ownership of farmers' land for chili planting ranges from 50 to 75 acres/farmers and planted tomato of 50 ha from 646 farmers. Ownership of farmers' land for chili is ranged between 15 -25 are / farmers. But there are still many obstacles that need to be fixed.
The constraints on the production of horticultural crops is the cultivation still needs to be improved on the three commodities, including fertilization, maintaining a healthy garden environment pest prevention, water, and market problems.
In addition, from the results of the study, it is expected that the development of horticultural crops in the village should be developed into finished products such as dodol, sweets, refreshments for drinks and sauces for chilies and tomatoes.


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Synthetic Grey Water Treatment Through FeCl3-Activated Carbon Obtained from Cotton Stalks and River Sand

The research objective was to reclaim greywater through simple, easily available, and cost-effective methods. For this purpose, an activated charcoal was prepared from biomass (cotton stalk) through the pyrolysis process and sand collected from river Indus. Both materials were subjected to separate columns and applied as filters. Whereas, the efficiency of both materials as filter media was analyzed on the synthetic grey water (SGW). The formulation of synthetic greywater was a complicated process because the selection of ingredients and their amount should not exceed from the real grey water. So, for the presence of fecal contamination, a small amount (10 ml L -1 ) of settled sewage was added to the distilled water, while to mimic the organic load, several chemical products of technical grade were also added. The physicochemical and microbiological characteristics of this SGW were tested before and after treatment. The results show that both mediums (AC and river sand) were very effective in the greywater treatment. The removal efficiency for BOD and COD was up to 91.2%, and 70% respectively. Similarly, the removal measure for turbidity was 91.3%. While the pH showed that the synthetic grey water was alkaline in nature with a value of 10 because the washing detergents used during the preparation of SGW, but after passing through both filter columns, pH was observed in between 7 and 8 units. Furthermore, the removal value examined after passing SGW from both columns for total coliforms was 46.87 CFU/100 ml from1500 CFU/100 ml.

Introduction
Dissolved pollutants (organic and inorganic) of water can be removed through an effective adsorption technique. Activated carbon (AC) is very familiar with all types of adsorbents due to high adsorption capacity. The adsorption frequency of activated charcoal relates to its great surface area, high pores distribution, and rapid grade of external reactivity [1]. Activated carbons are mostly extracted from raw carbon-rich resources in an oxygen-tight atmosphere through carbonization and followed by the activation process of the charcoaled material. The activation process can be carried out by means of chemical or physical activation [2]. Iron salts allow the preparation of activated carbons having a high specific surface area (965 m 2 g-1 ) and very small pores by activation at temperatures far below those used for activators generally described in the literature. Characterization studies have shown that the components present in the iron-impregnated material are completely pyrolyzed at a temperature of 280 °C [3]. Agro-industry produce millions of tons of lignocellulosic waste every year all over the world, additionally, usage of such wastes for activated carbon production will also help in reducing solid waste disposal issue [4]. Subsequently, an extensive range of agro wastes has been explored in Pakistan to produce activated carbon [5]. The cotton stalk is the fourth largest crop of Pakistan and every year about 25100 million tons of cotton stalk waste produced in the country [6]. The proximate analysis of the shredded cotton stalk in terms of bulk density 34.92 kg/m 3 , moisture content 13.63 %, volatile matter 74.52 %, ash content (4.95 %, fixed carbon 20.53 % and calorific value of cotton stalk biomass (3827 cals/g) respectively [7]. By utilizing cotton stalk for the preparation of activated carbon will provide an economical way for a beneficial product as well as it will also help the waste management issue. In addition, second media slow sand bed filter was also used to improve the efficiency of activated carbon treatment process because AC has some limitations on pathogen removal [8], while slow sand filters (SSF) can be easily obtained from local constituents and having an ability to remove 99% pathogens [9]. Moreover, water scarcity has developed a universal problem in current years. Now the water reclamation is the only way of preserving freshwater reservoirs. The interesting aspect is not to reclaim all household wastewaters but only grey waters (GW), this reclaimed water decreases potable water consumption by 29% to 47% [10]. Furthermore, Greywater is the whole domestic water arises from shower or bath, kitchen activities, and laundry purpose only excludes water containing excreta. It is the major point pollution source, which directly discharged from societies to the rivers and sea without any further treatment process [11]. There are several parameters such as quality and quantity of water supply, local practices, the culture of the area, and washing at the water source or home etc., influence the characteristics of greywater [12]. The most common method used for the physical treatment of greywater is recognized as filtration. While sedimentation or disinfection is a prior method applied to coarse filtration like sand bed filters. The highest rate for the removal of dissolved and suspended solids was obtained in membrane filtration but it had some limitations on organic pollutants [13]. The reason to select synthetic grey water (SGW) for the assessment of FeCl3-Activated carbon efficiency instead of real grey water (RGW), was the variation in the characteristics of real greywater [14] and recipe followed in the current study was a part of a previous research [15]. The objectives of this research were to study the effects of the salt based activating agent (Iron Chloride) in the production of activated carbon and to analyze its efficiency on the synthetic greywater. The temperature (400 0 C) and the volume of activating agent (4 th part of charcoal material) used in the activation process were at its minimum level, but the adsorbent (FeCl3-AC) obtained showed maximum level of efficiency during the treatment of synthetic greywater (SGW).

Collection, Modification and Gradation Analysis of River Sand
The Indus Delta River is an arid climate formed by high discharges from river sediments (about 400 million metric tons of sediment per year), moderate tidal range (2.62 m), and extremely high wave energy (14×107 erg/s). Coast and average wave height of 1.84 meters, strong wind monsoon in the southwestern part of summer and northeastern winter. The delta of the rather rough grain thus produced has a lobed shape, lacks dense vegetation, and is dissected by many tidal channels with mangroves in the lower delta plain. A prominent distribution in sedimentary basins, and a system dominated by waves, with little distribution along the coast unless it is characterized by sediments and sand dunes [16]. The sand used in this study was obtained from the lower delta plain, near Hyderabad, Sindh, Pakistan to fabricate the sand filter column. The obtained sand was washed thoroughly after collection in order to remove dirt and other impurities. Furthermore, after washing process, the residual quantity of sand was dried through an oven for 1 day at temperature of 110 0 C. The gradation analysis by wet sieving method used to analyze the grain size of sand [17].

Preparation and Characterization of FeCl3-Activated Carbon
The FeCl3 Activated charcoal was obtained from biomass (cotton stalk) through the pyrolysis process. The first stage was carbonization of biomass at 550 0 C for 1 hour. After carbonization charcoal was active with the help of an activating agent (iron III chloride or FeCl3) at 400 0 C. The mass ratio between the activating agent and charcoal was 1:4 respectively. Alert considerations regarding particle size distribution can provide substantial operational assistance. This allows the fixed contaminants to adsorb to the surface of the adsorbent. Representative particle size values for CA samples were achieved during the test. Use ASTM D-2862-97 (reapproved in 2004) [18].

Preparation of Synthetic Grey Water
Greywater is a type of wastewater used for various activities like washing purpose, kitchen, and showers etc. excluding excreta arise from toilets. Hair's traces food constituents, household products and dirt can be observed in the composition of greywater. It may even appear unclean, but at the same time in some cases it can be a valuable for plants. The availability of nutritious elements in greywater released from homes are the basic cause of pollution, and these nutritious elements can be a productive fertilizer for plants [19]. In this study, the synthetic greywater was prepared from components usually present in graywater. The constituents including 85 mg/L of dextrin, 75 mg/L of ammonium chloride, 70 mg/L of yeast extract, 55 mg/L of each starchy food and washing soda, 30 mg/L of washing powder, 11.5 mg/L of sodium dihydrogen phosphate, 4.5 mg/L of arcanite, 10 ml/L of settled sewage and 0.1 ml/L of each shampoo and oil as shown in Table 1. The initial characterization of synthetic greywater was analyzed on 5 liters, while for the final application of iron chloride based-AC in combination with sand bed filter 30 liters of synthetic greywater was prepared. The recipe of synthetic greywater used in this study to analyze the efficiency FeCl3-AC and sand bed filter was nearly of the same nature as real grey water [20].

Fabrication of FeCl3-Activated Carbon Filter and Slow Sand Bed Filter Columns
In this regard two columns of transparent material having length 40 cm and 8 cm diameter were installed in vertical series, holding AC filter on the upside and sand filter on the downside. A locally available fabric was also used at the lower ends of both columns to separate the mixing of materials filled and hold them from the drain. Furthermore, both columns were connected to a sedimentation tank having a capacity of 30 liters. The sedimentation time for SGW was 3 hours before it flows towards filters. AC and sand columns were nearly filled up to 25 cm [21].

Characterization of Synthetic Grey Water
The structure of the synthetic grey water was reconstructed in the present study that is shown in Table 3. This is largely composed of household products including organic and inorganic pollutants from bathrooms, kitchen, and laundry. The characteristics of synthetic greywater were observed in this study were biochemical oxygen demand (BOD), chemical oxygen demand (COD), Turbidity, the potential of hydrogen (pH), and Total coliforms [22]. Aerobic bacteria utilize free oxygen for the decomposition of organic food present in wastewater and BOD is a valuation of that organic food. The consumptions of DO in wastewater is directly proportional to the amount of organic food presence. This test investigates the oxygen usage by bacteria needed them for stabilization of organic matter under measured environments. The BOD of the sample was measured through the titration method. In this assay DO initial was measured on the first day, and after five days DO final was measured. The difference between DO1 and DO5 was the BOD5. COD is the measure of the whole quantity of oxygen required for biological life and the decomposition of organic food. Chemical oxygen demand (COD) is basically the valuation of oxygen needed for the oxidation process of organic material into CO2 and H2O. The method used for the COD was closed reflex calorimetric method. Turbidity is the examination of comparative clearness of liquid. Turbidity is an appearance of the light scattered when it passes through the water. More the light dispersed, higher will be the turbidity value. Several impurities like clay, silt, algae, microscopic organisms, fine particles of organic and inorganic compounds. The equipment used to measure the amount of turbidity in synthetic greywater before and after the application of filter mediums was turbidity meter. The degree of acidity and basicity of a solution is known as pH. The value of the pH scale is divided into 14 figures, while 7 is the neutral point represent water. Above 7 all are alkaline and below 7 all are acidic in nature. The parameter was measured through pH meter. Total coliform calculation suggests an overall sign of the disorder of water. The more the value, the higher the contamination level of water. While total coliforms can originate from sources other than feces. The method used to analyse the measuring values of total coliforms at an initial and final stages in synthetic greywater was total coliforms colonies technique through membrane filtration.

Flowchart
Flowchart of the research methodology has been presented as bellow:

Gradation Analysis of River Sand by Wet Sieving
The sieve analysis of collected sand identify that the distribution of particle size for river sand was 0.075 mm (see Table 2). While the basic purpose to use sand filter column as an additional filter medium with activated carbon was to treat the amount of total coliforms may appears in synthetic greywater. The particle size of sand was the most prominent parameter in this consideration. A significant interaction between filter depth and the flow-through rate in the removal of E. coli (p ˂ 0.05) was observed which means that increasing the depth of the sand filter while slowing the filtration rate improved efficiency in E. coli removal of the raw water [23].

Determination of Particle Size of Powdered Activated Carbon
A particle size distribution caution can provide substantial operational assistance. This allows you to fix the adsorption of dirt on the surface of the adsorbents. During testing, typical particle sizes of the activated carbon was achieved. Though ASTM D-2862-97 (approved in 2004), 50 g of AC were investigated, while the main part of 50 g was stored in the opening of the screen 1.44 mm, moreover, the screen was opened 3.57 and 2.03 mm has 11.2 and 12.4 g, respectively, as shown in Table 3. Activated carbon with a particle size of less than about 0.4 mm is considered to be a poor filtering medium and is processed for sale in powder form [24].

Analysis of Synthetic Grey Water before and After Treatment with FeCl3-AC and Slow Sand Bed Filter
A noticeable effect in quality of distilled water was examined after the addition of ingredients used to prepare the synthetic greywater. The synthetic greywater was tested several times at an initial and final stages in order to obtain the accurate results. The sedimentation time provided to synthetic greywater was 3 hours. After sedimentation effluent synthetic greywater flows to wards activated carbon and sand filter in a continuous stream. The SGW retention time for any column is zero and the two filters are proven to be efficiencies up to 60 liters. In addition, continuous changes were observed in all SGW parameters due to the addition of 10 ml/l of precipitated Sewage 2. The weight of the ingredients used to make the SGW is slightly different. Significantly changed the value of BOD5. The SGW values at an initial stage before the treatment were 15.2-40.6 mg/liter, and the results after applying the filter were 1.06-3.26, as shown in Table  4. The BOD5 removal rate of activated carbon and sand bed filter can be 52.41 to 5.36 mg/liter [22].

Figure 5. BOD5 of synthetic grey water before and after treatment
The COD measures the value of the synthetic grey water before the treatment is above the BOD because many organic materials are chemically oxidized rather than biological. Before using the river sand filter and activated carbon, the COD value was 48-81 mg/l, and after application, the value was between 21.4-23.8 cm. Table 4. A pervious study noted that the COD reduction values were almost equal, the percentage exclusion rate was 65%, and the percentage reduction ranged from 54% to 70% [21].

Figure 6. COD of synthetic grey water before and after treatment
Greywater can be composed of several contaminants which can affect through various ways. Turbidity is the parameter used to identify the presence of these complex nature pollutants. The turbidity of the finished grey water changes during the early stages prior to treatment. The measured value before treatment is 28.3-36.8 NTU, and the measured value after filtration is 1.93-3.2. A study showed that the use of filter sand and activated carbon keeps the turbidity below 4 NTU [13]. 25  Due to the detergent used to make it, the properties of synthetic grey water are very strong alkaline. The pH of grey water (5-10.9) is more alkaline than the common household wastewater (5.9-7.7) [25]. The initial pH was observed between 9.2 and 10 and after filtration with FeCl3-Based activated carbon in combination with sand filter, the range was 7 to 8. The pH results were consistent with previous studies between 7.2 and 7.9 after treatment [26].

Figure 8. pH of synthetic grey water before and after treatment
Literature identified that the total coliforms in clear water ranging from 500 to 2.4 × 10 7 CFU/100 ml [27]. In this research, the initial results of total coliforms in synthetic grey water was as high as 900-1500 CFU/100 ml, and even the amount of precipitated sewage mixed to SGW was only 10 ml/l, but the effect was significant. After applying the both filters i.e. iron chloride based Ac and sand filter, the final measuring values range were 6.32 to 46.87 CFU/100 ml, which clearly excludes the total SGW.

Conclusion
Synthetic grey water was synthesized, and the effectiveness of AC in local agricultural waste and the effect of sand filter combined with AC in SGW were analyzed. The ingredients used to make the SGW are the main components of domestic products. The research analyzed that the combined filters (AC and river sand) were very operational in treating grey water. Compared with BOD, synthetic grey water has a higher COD value, but the combined elimination of the two filters is more efficient than COD. In addition, activated carbon and river sand have proven to be very effective at the pH of the synthetic grey water used for treatment, which is highly alkaline. During the treatment, the average removal efficiency of turbidity was analyzed to be 91.3%. Although the overall elimination rate of E. coli was also high, the average percentage was 97.9%. The total efficiency of activated carbon and river sand filters based on FeCl3 is better, but the retention times of both columns are zero, and the retention time of SGW in both columns is studied to provide better results

Acknowledgment
Authors would like to thank Dr. Khan Muhammad Brohi Dean faculty Architecture and Civil Mehran University of Engineering and Technology Jamshoro, Sindh, Pakistan for the direct involvement during the initial laboratory trials and continuous trial. [2] Yakout SM, Sharaf El-Deen G. "Characterization of activated carbon prepared by phosphoric acid activation of olive stones." Arabian Journal of Chemistry 9 (Nov 2016): S1155-S1162. doi:10.1016/j.arabjc.2011.12.002.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Evaluation of Bacterial Endophytes for their Plant Growth Promotion in vitro Condition

Endophytes are the group of microbes may be fungi or bacteria which asymptomatically inhabit the internal tissues of plants; they colonize the same ecological niches as disease-causing organisms. Endophytes are are known to control the plant diseases by suppressing the pathogens but they also helps in plant growth promotion. Some of the mechanisms in plant growth promotion are biological nitrogen fixation, ACC deaminase synthesis, siderophore production, IAA production, phosphate solubilization, induction of host plant resistance mechanisms, phytohormone production and synthesis of pathogen inhibiting volatile compounds (Glick, 2015). The seed play an important role in agriculture . The overall development of the plant depends upon the seed germination and vigour of seeds. Malfanova et al., (2011) tested the plant growth promoting abilities of the bacterial endophytes in tomato, wherein he isolated 30 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 04 (2019) Journal homepage: http://www.ijcmas.com

Introduction
Endophytes are the group of microbes may be fungi or bacteria which asymptomatically inhabit the internal tissues of plants; they colonize the same ecological niches as disease-causing organisms. Endophytes are are known to control the plant diseases by suppressing the pathogens but they also helps in plant growth promotion. Some of the mechanisms in plant growth promotion are biological nitrogen fixation, ACC deaminase synthesis, siderophore production, IAA production, phosphate solubilization, induction of host plant resistance mechanisms, phytohormone production and synthesis of pathogen inhibiting volatile compounds (Glick, 2015). The seed play an important role in agriculture . The overall development of the plant depends upon the seed germination and vigour of seeds. Malfanova et al., (2011) tested the plant growth promoting abilities of the bacterial endophytes in tomato, wherein he isolated 30 Endophytes are the micro-organism which shares the life long relation with the host. The bacterial endophytes promote the growth directly and indirectly by several mechanisms. Keeping in mind, the present investigation has been carried out to evaluate the bacterial endophytes for their plant growth promotion in vitro condition. Four screened bacterial antagonists viz., SVC 11, BE 1, NGB 21 and M1W1 were evaluated for the germination and the vigour of French bean, brinjal and rapeseed mustard seeds. The bacteria were used individual as well as in consortium mode on the respective seeds. The result revealed that the seeds of French bean and brinjal treated with isolate NGB 21 as best in terms of germination (91.11% and 84.44% respectively) and vigour (3270.88 and 1094.96 respectively) followed by BE 1. Isolate SVC 11 showed the highest vigour (630.51) at 21st day and germination (84.44%) followed by the isolates NGB 21 and BE 1 in rapeseed mustard. The root length and shoot length were significantly higher in French bean as compared to brinjal and rapeseed mustard. Statistically the germination percentage and the vigour index were found to be significant. The result showed that all the screened potential bacteria have the best plant growth promoting ability as compared to the other crop seed tested. Hence, the microbial consortia could be evaluated further under field conditions to developed consortia against major vegetable pathogens of Meghalaya.
bacterial endophytes from different plant species and evaluated against tomato foot and root rot (TFRR). Of which, B. subtilis (HC8), which was isolated from the giant hogweed, significantly promoted the plant growth along with giving protection against TFRR.
He indicated the production of metabolites like gibberellin hormone, lipopeptide antibiotics as possible mechanism for plant growth promotion and disease suppression respectively. Wahyudi et al., (2011) reported the ability of Bacillus sp. to root length, shoot length of seedlings (seed germination biosssay) in vitro. Agrawal and Agrawal, (2013) also isolated Bacillus strains from the rhizosphere of tomato plants and screened for their plant growth promoting activities. Five isolates were selected and they showed increased shoot and root length as well as increased seed germination and seedling vigour. The ability of Bacillus spp. on the growth promotion in-vitro has been reported by several workers (Wahyudi et al., 2011;Agrawal and Agrawal, 2013;Malleshwari et al., 2013)

Inoculam preparation
Four endophytic bacteria used in this study (SVC 11, BE 1, NGB 21 AND M1W1 ) were isolated in the laboratory from different plant tissues and stored at -80 °C in 20% (v/v) glycerol. To prepare inoculam, 1 ml aliquot of a bacterial culture grown overnight (approximately OD600=1.0) were inoculated in 20 ml nutrient broth (3 g beef extract, 10 g proteose peptone, 5 g NaCl in one liter of dH2O, pH 7.2) at 30 °C for 24 h with 150 rpm shaking in rotary shaker. The individual isolates were subjested for compatibilty test and the microbial consortia was prepared by mixing the bacterial growth in the aliquot.

Germination assay
Germination assay was performed on the petri dishes (150mm diameter). The seed is surface sterilized with 0.1% mercuric chloride for 3 min and washed with distilled water for 4-5 times. The seeds were soaked for 3h in 48 h old bacterial broth cultures containing at least 10 -6 cells/ml and placed in the Petri dish containing the paper towel moistened with water at the rate of 15 seeds per plate and replicated 3 times. One treatment was carried out by mixing the 4 bacterial cultures (Microbial consortium). The seeds treated with sterile water were served as control. The plates were incubated at 28 ± 2 o C and moisture of the petri dishes was maintained regularly.
Selection-9 variety of beans, KSP-1164 Devansh variety of brinjal and variety of Rapeseed Mustard were used for the present investigation. The observations for percentage seed germination were taken at 7 days after incubation. The observations for root, length, shoot length and vigour length was taken at 7, 14 and 21 days after incubation.

Experimental design and Statistical analysis
The above experiment was designed in completely randomized design (CRD).The data were analyzed using one-way analysis of variance (ANOVA). Statistical significance between the treatments was compared using LSD (Least significant difference) and a P <0.01 was considered as significantly different from the other treatments (Gomez and Gomez, 1984).

Results and Discussion
PGPB may affect plant growth either directly or indirectly. The attributes contributing to the promotion of plant growth includes production of phytohormones like IAA (Pattern and Glick, 1996), Giberrelic acid (Mahmoud et al., 1984), Cytokinin (Tien et al., 1979), production of ACC deaminase enzyme, nitrogen fixation, solubilisation of phosphate. The ability of Bacillus spp. on the growth promotion in-vitro has been reported by several workers (Wahyudi et al., 2011;Agrawal and Agrawal, 2013;Malleshwari et al., 2013). Four screened bacterial antagonists were subjected for the compatibility among themselves and n o lysis was observed at the juncture of SVC 11, BE 1, NGB 21 and M1W1 combinations, which indicated the compatibility among the isolates. It was found that seeds of French bean treated with isolate NGB 21 recorded as best in terms of germination (91.11%) and vigour (3270.88 atv 21 st day) followed by M1W1 (85.22% and 2809.25, germination percentage and vigour index respectively) and BE 1 (82.22% and 2864.07, germination percentage and vigour index respectively) (FIg. 1 and 2; Table 1-3).     Table.3 Evaluation of screened isolates for PGP activities in rapeseed mustard seeds In vitro    In conclusion, isolate NGB 21 as best in terms of germination and vigour. All the screened potential bacteria has the best plant growth promoting ability as compared to the other crop seed tested. Hence, the microbial consortia could be evaluated further under field conditions to developed consortia against major vegetable pathogens of Meghalaya.