New horizon to the world of gut microbiome: seeds germination.

The second brain of humans has been known as the microbiome. The microbiome is a dynamic network composed of commensal bacteria, archaea, viruses, and fungi colonized in the human gastrointestinal tract. They play a vital role in human health by metabolizing components, maturation of the immune system, and taking part in the treatment of various diseases. Two important factors that can affect the gut microbiome's composition and/or function are the food matrix and methods of food processing. Based on scientific research, the consumption of whole grains can make positive changes in the gut microbiota. Seeds contain different microbiota-accessible substrates that can resist digestion in the upper gastrointestinal tract. Seed germination is one of the simplest and newest food processing approaches to improve seeds' bioavailability and overall nutritional value. During germination, the dormant hydrolytic seed's enzymes have been activated and then metabolize the macromolecules. The quality and quantity of bioactive compounds like prebiotics, fiber, phenolic compounds (PC), total free amino acids, and γ-aminobutyric acid (GABA) can increase even up to 4-10 folds in some cases. These components stimulate the survival and growth of healthful bacteria like probiotics and boost their activity. This effect depends on several parameters, e.g., germination environmental conditions. This review aims to provide up-to-date and latest research about promoting bioactive components during seed germination and investigating their impacts on gut microbiota to understand the possible direct and indirect effects of seed germination on the microbiome and human health.

Gut microbiome plays a vital role in human health.Promoting gut beneficial bacteria can treat some human diseases.Beneficial gut bacteria can improve by seed's bioactive compoundsSeed's bioactive compounds can increase during germination.Germination is a low-in-cost process that can make an indirect positive effect on the gut.

Mixed fermentation and electrospray drying for the development of a novel stabilized wheat germ powder containing highly viable probiotic cultures.

Nondairy fermented probiotic powder was developed based on stabilized wheat germ through mixed fermentation (Lactobacillus acidophilus and Lactobacillus plantarum) and electrospraying process. In the first step, the effect of mixed fermentation on lipase and lipoxygenase activity of wheat germ was investigated. The results showed a significant reduction in the activity of both enzymes (82.72% for lipase and 72% for lipoxygenase), therefore, mixed fermentation effectively stabilizes the wheat germ. In the next step, after the preparation of the solutions for drying process and investigating the physical properties (surface tension, electrical conductivity, and viscosity) of the solutions, the electrosprayability of the samples was evaluated at different conditions and revealed that 18 kV applying voltage, 0.3 flow rate, and 12 cm distance between tip to collector was the best for electrospraying the 20% solution of fermented wheat germ with morphologically most semi-uniform particles. Finally, the viability of the probiotics after drying process and during the storage at 25°C was examined. The number of initial cells counted as 14.48 ± 0.2 log cfu/g and the viability studies showed 0.55 log cfu/g decrease in the number of viable bacteria from initial count as a result of the electrospraying process. Furthermore, 7.86 ± 0.03 log cfu/g in freeze-dried and 9.05 ± 0.45 log cfu/g in electrosprayed samples survived after 70 days of storage.

Diabetes and seeds: New horizon to promote human nutrition and anti-diabetics compounds in grains by germination.

Type 2 diabetes (T2D) is a complex and heterogeneous chronic metabolic disorder disease that is associated with high blood sugar. Because of the side effects of synthetic drugs on T2D patients and their economic burden, interest in plant-derived functional foods like grains with biological activities has developed. Based on scientific reports, whole grains are rich sources of energy, nutrients, and bioactive compounds and are assumed to have beneficial health effects on glucose enzymes regulation or hyperglycemia. Nowadays, different methods have been applied to enhance whole seed healthful properties and anti-diabetic compounds, and germination is one of them. Germination (sprouting) is a cost-effective method for boosting the activity of endogenous seed enzymes and modifying the structure of macromolecules. Some of these macromolecules like bioactive peptides, polyphenols, dietary fiber, and vitamins are related to diabetes management. Determining the best germination condition can help to promote these anti-diabetics properties of compounds. This study presents relevant information about diabetes, the effect of seed germination on releasing bioactive compounds, and optimizing environmental germination conditions to improve the anti-diabetic compounds in seeds for reaching functional food.

Tuning the Physicochemical, Structural, and Antimicrobial Attributes of Whey-Based Poly (L-Lactic Acid) (PLLA) Films by Chitosan Nanoparticles.

Recently, the research and innovation to produce raw materials from microbial processes has gained much attention due to their economic and environmental impacts. Lactic acid is a very important microbial product due to its wide application in the food, pharmaceutical, cosmetic, and chemical industries. In the current study, poly (L-lactic acid) (PLLA) was produced by the ring opening polymerization (ROP) technique of L-lactic acid recovered from whey fermentation, and was used for the production of nanocomposites films reinforced with chitosan nanoparticles (CNPs) (average diameter ca. 100-200 nm). Three different CNPs concentrations, namely 1, 3, and 5% w/w, were tested, and their influence on the physical, mechanical, thermal, antibacterial and structural attributes of PLLA film was assessed. The results showed that the addition of CNPs up to 3% caused a significant improvement in water vapor permeability, appearance, tensile strength and elongation at break. The antibacterial properties of nanocomposites followed a dose-depended pattern as a result of CNPs addition. Therefore, the best inhibitory effects on Escherichia coli and Staphylococcus aureus was made by the addition of 5% of CNPs and lower dosages slightly affected the growth of pathogens or didn't cause any inhibitory effects (in 1% of CNPs). It can be concluded that the incorporation of CNPs into the PLLA matrix allows to improve the structural, thermal, physical, mechanical and antibacterial properties of the polymer, generating promising systems for food packaging and biomedical applications.

Canthaxanthin Biofabrication, Loading in Green Phospholipid Vesicles and Evaluation of In Vitro Protection of Cells and Promotion of Their Monolayer Regeneration.

In the present study, canthaxanthin was produced by biofermentation from Dietzia natronolimnaea HS-1 (D. natronolimnaea) and was loaded in phospholipid vesicles prepared with natural component using an easy and low dissipative method. Indeed, glycerosomes, hyalurosomes, and glycerohyalurosomes were prepared by direct hydration of both phosphatidylcholine and the biotechnological canthaxanthin, avoiding the use of organic solvents. Vesicles were sized from 63 nm to 87 nm and highly negatively charged. They entrapped a high number of the biomolecules and were stable on storage. Canthaxanthin-loaded vesicles incubated with fibroblasts did not affect their viability, proving to be highly biocompatible and capable of inhibiting the death of fibroblasts stressed with hydrogen peroxide. They reduced the nitric oxide expression in macrophages treated with lipopolysaccharides. Moreover, they favoured the cell migration in an in vitro lesion model. Results confirmed the health-promoting potential of canthaxanthin in skin cells, which is potentiated by its suitable loading in phospholipid vesicles, thus suggesting the possible use of these natural bioformulations in both skin protection and regeneration, thanks to the potent antioxidant, anti-inflammatory and antiageing effects of canthaxanthin.

Economic purification of recombinant uricase by artificial oil bodies.

Rasburicase is an expensive treatment used to control hyperuricemia caused by tumour lysis syndrome (TLS). In this study, a non-chromatographic method was designed based on nano-oil bodies for convenient and economical purification of the recombinant uricase. For this purpose, two chimaeras were synthesized with a different arrangement of the uricase, caleosin and intein fragments. After confirming the protein expression by measuring the uricase activity at 293 nm, purification was conducted through oil-body construction. The results were resolved on the 12% SDS-PAGE gel. Finally, the stability of the oil bodies was examined against different salts, surfactants, temperatures, and pH values. According to our results, the overexpression of uricase-caleosin chimaera under the T7 promoter in Escherichia coli led to the production of soluble protein, which was successfully purified by artificial oil bodies. The active uricase was subsequently released through the self-splicing of intein. Further investigations highlighted the importance of the free C-terminus of caleosin in constructing artificial oil bodies. Moreover, surfactants and low temperature, in contrast to salts, improved the stability of oil bodies. In conclusion, caleosins are an efficient purification tag reducing the cost of purification compared to conventional chromatography methods.

Investigation of the possibility of fermentation of red grape juice and rice flour by Lactobacillus plantarum and Lactobacillus casei.

The aim of the current study was to evaluate the possibility of the bacterial growth and substrate metabolism during the fermentation of red grape juice and the mixture of red grape juice and rice flour solution using Lactobacillus plantarum and Lactobacillus casei. In recent years, cereal-based beverages have been used as functional compounds such as antioxidants, dietary fiber, minerals, probiotics, and vitamins in diets. In this research, fermentation of red grape juice (media 1) and 1:1 mixture of red grape juice and rice flour solution (media 2) by two strains of gram positive and homofermentative lactic acid bacteria: L. plantarum and L. casei (individually and mixed) was examined. Fermentation was carried out at 37°C for 48 hr. Microbial population, pH, acidity, sugar, and organic acid metabolism were measured during the fermentation period. Data showed that in media 2 fermented with mixed culture of both L. plantarum and L. casei, acidity and microbial population increased sharply at the initial stages of fermentation, and the most percentage of lactic acid production occurred. Red grape juice fermented with mixture of L. plantarum and L. casei showed the most sugar consumption (p < .05). Results indicated that the use of the mixture of red grape juice and rice flour solution can be a proper substrate for producing lactic acid.

Pulses' germination and fermentation: Two bioprocessing against hypertension by releasing ACE inhibitory peptides.

Angiotensin-Converting Enzyme (ACE) is one of the main blood pressure regulators in the renin-angiotensin system leading to hypertension. Hypertension is known as the modern world disease which increases the risk of serious human health problems. Synthetic drugs and some natural compounds could treat this disease by binding to ACE and reducing its activity. Pulses, one of the legumes group, that are the rich in protein sources in the human diet, have several bioactive compounds with ACE inhibitory (ACE I) properties. However, several processes need to break down proteins and improve ACE I activity in foods. Germination and fermentation, known by bioprocessing, could make releasing bioactive peptides and polyphenols and exhibit ACE I and either other health properties such as antimicrobial, antioxidant, anti-diabetic and anticancer activities. Various factors such as kind of selective culture, temperature, time and humidity affect these processes. This review summarizes relevant studies about the effect of pulses' germination and fermentation to produce ACE I activity compounds and also explains about main parameters affecting the health properties of these two bioprocessing to treat hypertension that could lead to the development of their application in pharmaceuticals instead of synthetic drugs.

Microencapsulation of microbial canthaxanthin with alginate and high methoxyl pectin and evaluation the release properties in neutral and acidic condition.

Canthaxanthin (CX) is an orange-red keto-carotenoid with high antioxidant activity. This functional pigment is sensitive to oxygen, light, pH and heat. In this study, CX was produced by the Dietzia natronolimnaea HS-1 and was encapsulated in Alginate (Alg) and Alg-high methoxyl pectin (HMP) through O/W/O multiple emulsion/external gelation method to developed resistant microparticles among acidic and neutral pHs. Results showed that initial CX concentration had a significant influence on total CX (TCX), surface CX (SCX), microencapsulation efficiency (EE) and particles size. The highest EE% for Alg (60.21 ±â€¯0.18) and Alg-HMP (70.60 ±â€¯0.68) were obtained with CX initial concentration of 11 and 18 µg/mg, respectively. Alg microparticles showed smaller size compare to Alg-HMP microcapsules. Presence of CX in microparticles and good antioxidant activity was confirmed by FT-IR spectroscopy and DPPH assay, respectively. CX in vitro release was 66% and 49% in acidic condition and 76% and 50% in neutral condition for Alg and Alg-HMP, respectively. Thus, Alg-HMP-CX18 microparticles were selected to be used in both neutral and acidic foods such as milk and fermented milks products as an antioxidant and a colorant agent.

Optimization of Effective Minerals on Riboflavin Production by Bacillus subtilis subsp. subtilis ATCC 6051 Using Statistical Designs.

BACKGROUND: Riboflavin (vitamin B2) is an essential component of the basic metabolism, and an important nutritional and growth factor in humans, animals, plants and micro-organisms. It has been widely used in the fields of pharmaceuticals, feed and food additives. The industrial production of riboflavin mostly relies on the microbial fermentation. Designing an appropriate fermentation medium is of crucial importance to improve the riboflavin production. METHODS: In this study, sequential methodology combining a screening test of minerals by Plackett-Burman (PB) and an optimization test by Central Composite Design (CCD) was applied to enhance riboflavin production by Bacillus subtilis ATCC 6051 in shake flasks. RESULTS: Initially, one-factor-at-a-time approach was applied to evaluate the effect of different carbon sources. The results showed that fructose was significantly most effective on biomass and riboflavin production. After that, 13 minerals [CaCl2, CuCl, FeCl3, FeSO4, AlCl3, Na3MoO4, Co(NO3)2, NaCl, KH2PO4, K2HPO4, MgSO4, ZnSO4, and MnSO4] were studied with the screening test. The results revealed that concentration of MgSO4, K2HPO4, and FeSO4 had greater influence on riboflavin production (p< 0.05). A CCD with five factors (concentration of fructose, MgSO4, K2HPO4, FeSO4, and yeast extract) at five levels was then used to determine the maximum riboflavin concentration. The optimal concentrations (g/l) of these variables determined by Response Surface Methodology (RSM) were fructose, 38.10; MgSO4, 0.85; K2HPO4, 2.27; FeSO4, 0.02; and yeast extract, 4.37. CONCLUSION: Statistical experimental design offers a practicable approach to the implementation of medium optimization. From an industrial view point, our optimum medium, besides fructose and a small amount of yeast extract, is mainly composed of common and cheap inorganic salts, which are available to the industrial riboflavin production.

A comparison of Canthaxanthine Pickering emulsions, stabilized with cellulose nanocrystals of different origins.

Cellulosic nanocrystals from different origins were made to stabilize the canthaxanthin (CTX) in pickering emulsion. Nanocrystals were obtained by hydrochloric acid hydrolysis. Dynamic light scattering (DLS) demonstrated that the length of solid particles were in the range of 112nm-4000nm. AFM indicated the needle-like shape of the cotton cellulose nanocrystals (CCNs) and bacterial cellulose nanocrystals (BCNs) and also illustrated the thickness of the particles to be 6 and 7nm respectively. The crystallinity of both BCNs and CCNs was higher than the cellulose source they were isolated (CL: 75.4%, CCNs: 86.6; BC: 79.2%, BCNs: 88.5%). Effects of different factors such as temperature, pH and ionic concentration on stability were investigated. The results revealed that an increase in both temperature and pH was accompanied by an improvement in emulsion stability in all tested samples. However, increase in ionic concentration resulted in emulsions with less stability. In all the samples, CCN emulsions had better stability than the BCN emulsions, which was associated to smaller particle size and more coverage ability. Test of stability to light showed that CCN emulsions can preserve CTX better than BCN against the light.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Improving the integrity of natural biopolymer films used in food packaging by crosslinking approach: A review.

Currently used approaches for biopolymer modification are either expensive, poisonous or do not lead into the well-desired characteristics to the final film materials. Development of crosslinking procedure is an innovative strategy to improve mechanical, physical and thermal properties of biopolymer films. This review provides a brief description of film-forming biopolymers (e.g. chitosan, whey protein, alginate and starch) followed by a detailed introduction to definition and classification of various crosslinkers, the effect of crosslinking on emerging attributes of biopolymer films including physical, mechanical and thermal properties, identification of crosslinking occurrence, and cytotoxicity status of commonly used crosslinkers in the field of food and food-related packaging materials.

Nanoparticles based on crocin loaded chitosan-alginate biopolymers: Antioxidant activities, bioavailability and anticancer properties.

Crocin, a water-soluble carotenoid in saffron, is a highly bioactive compound with limited use due to instability to pH variations, heat and oxidative stress, rapid absorption and low bioavailability. In this study, chitosan-alginate nanoparticles, considered as a new vehicle for crocin to improve properties in terms of antioxidant activity, bioavailability and anticancer activity. The swelling of crocin loaded nanoparticles was pH-dependent so that the highest swelling ratio (16g/g) was observed in acidic condition (pH=1.2) after 24h. The antioxidant activity of the crocin loaded nanoparticles was the same effective as pure crocin on DPPH free radical scavenging and ferric reducing ability of plasma (FRAP). In vitro crocin release profile from composite nanoparticles was investigated under simulated physiological conditions during incubation time. The experimental reports collected in this study strongly indicate that nanoparticles loaded with crocin could be the prospective candidates for the future anti-cancer therapeutics that deserve additional investigations.

Proniosomal powders of natural canthaxanthin: Preparation and characterization.

In this study, canthaxanthin (CTX) was produced by Dietzia natronolimnaea HS-1 using beetroot molasses as substrate and used for encapsulation in proniosome powders after extraction, with the aim of improving its stability. Proniosome powders were prepared with an equimolar ratio of span 60/cholesterol and four different carriers, namely maltodextrin, mannitol, lactose and pullulan. The properties of these formulations as both proniosomal powders and resulted niosomal dispersions were evaluated. The type of carriers had significant effects on the micrometric properties of proniosome powders which were further confirmed by the results of SEM analysis. Although light and high temperatures affected the stability of CTX drastically, but encapsulation in proniosomes retarded its degradation. Among these samples, mannitol based proniosome powder (MAPP) produced small vesicles (mean diameter=175±3nmand polydispersity index=0.28±0.02) with the highest entrapment efficiency (74.1±2.7%). MAPP provided a promising formulation to increase CTX stability especially upon storage at high temperatures (45°C).

Optimization of crosslinked poly(vinyl alcohol) nanocomposite films for mechanical properties.

The effects of glycerol, bacterial cellulose nanocrystal (BCNC) and boric acid concentrations on the mechanical properties of PVA based films, including ultimate tensile strength (UTS), elongation at break (EAB), tensile Young's modulus (TYM), tensile toughness to break (TT), ultimate puncture strength (UPS), puncture deformation (PD), puncture Young's modulus (PYM) and puncture toughness to break (PT), were scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R2 values ranging from 0.945 to 0.977 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum UTS (72.84MPa), EAB (293.43%), UPS (4.64MPa) and PD (31.80%) could be achieved at 13.89% glycerol concentration, 5.00% BCNC concentration and a boric acid content of 1.96%. The predicted values for optimum conditions were in good agreement with experimental data. Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the formation of intramolecular and intermolecular hydrogen and ether crosslinkages in PVA and/or BCNC chains when boric acid is applied. Results showed that PVA/BCNC nanocomposite films plasticized with glycerol and crosslinked with boric acid showed appropriate mechanical properties that made them suitable as a disposable packaging film.

Polysaccharide type and concentration affect nanocomplex formation in associative mixture with ß-lactoglobulin.

The main objective of this work was to investigate the effects of polysaccharide type (gum Arabic, GA and/or carboxymethyl cellulose, CMC) and concentration (0-0.2% (w/w)) on the electrostatic interactions with ß-lactoglobulin (BLG). Biopolymer interaction was studied using turbidimetric analysis, dynamic light scattering (DLS), electrophoretic mobility and relative viscosity measurements. Complexes were developed at pH values below the isoelectric point (pI) of BLG. Increasing polysaccharide concentration increased the stability of nanocomplexes through increasing the negative charges carried by complexes. The differences between GA and CMC were attributed to the polysaccharide structure (linear or branched chains), charge density and flexibility. Phase contrast optical microscopy showed that complexation was of nucleation and growth type mechanism. Relative viscosity measurements confirmed that wrapping phenomenon occurred in both associative mixtures.

Effect of Lactobacillus casei- casei and Lactobacillus reuteri on acrylamide formation in flat bread and Bread roll.

The aim of this study was the evaluation of fermentation by lactic acid bacteria (LAB) contains lactobacillus (L.) casei- casei and L. reuteri on acrylamide formation and physicochemical properties of the Iranian flat bread named, Sangak, and Bread roll. Sangak and Bread roll were made with whole and white wheat flour, respectively. Whole-wheat flour had upper content of protein, sugar, ash, fiber, damaged starch and the activity of amylase than the white wheat flour. After 24 h of fermentation, the pH values of the sourdoughs made from whole-wheat flour (3.00, 2.90) were lower, in compared to sourdoughs prepared from white wheat flour (3.60, 3.58). In addition, in Sangak bread, glucose, and fructose were completely utilized after fermentation, but in bread roll, the reduced sugar levels increased after fermentation and baking that represent microorganisms cannot be activated and utilized sugars. Acrylamide formation was impacted by pH of sourdough and total reducing sugar (r = 0.915, r = 0.885 respectively). Bread roll and Sangak bread were fermented by L. casei- casei contained lowest acrylamide content, in two bread types (219.1, 104.3 µg/kg respectively). As an important result, the acrylamide content of Sangak bread in all cases was lower than in the Bread roll.

Improvement of crocin stability by biodegradeble nanoparticles of chitosan-alginate.

This study aimed to improve the stability of crocin, a saffron carotenoid, encapsulating into chitosan (Cs)-sodium alginate (Alg) nanoparticles prepared by a modified ionic gelation method were investigated as a new carrier to improve the stability of crocin. Response surface methodology was used to optimize the important variables, namely the concentrations of Cs and Alg, and pH influencing the particle size, zeta-potential, and encapsulation efficiency to find the optimum formulation for production of crocin nanoparticles (CNPs). Microscopic analysis and dynamic light scattering examination indicated non-smooth and spherical nanoparticles with the size range of 165-230 nm in weight ratio of Cs:Alg (1:1.25) and pH 4.7. Fourier transform-infrared spectroscopy displayed an extensive hydrogen bonding interaction between the crocin and biopolymers. Encapsulation efficiency, loading capacity and yield of CNPs were 38.16, 30.96 and 48.33%, respectively. The zeta-potential of NPs was about -33.52 mV which resulted in the better stability of NPs during manipulation and storage. Stability studies showed that nanoencapsulation provided enhanced crocin stability with biopolymers compared to the standard crocin under unfavorable environmental conditions.

Influence of supplemented diet with Pediococcus acidilactici on non-specific immunity and stress indicators in green terror (Aequidens rivulatus) during hypoxia.

Green terror fish were fed for two months with three types of feed including: control diet (C), diet containing 2% fish oil (O) and diet containing fish oil supplemented with 0.1% Pediococcus acidilactici bacteria (PA). At the end of the feeding period, 50 fish of different groups (n = 3) with an average weight of 4.28 g were transferred to 9 tank. Hypoxia tests were set out by sampling at three times including before hypoxia (BH) initiation, hypoxia stress spot (H) and starting mortality (SM). In fish submitted to the diet containing P. acidilactici bacteria, the immune indices of lysozyme activity (4.08, 4.19 and 4.85 µg/ml)], complement activity (2.65, 2.77 and 2.1 U/ml) and total immunoglobulin (10.05, 10.25 and 9.9 µg/ml) improved in all stages of sampling (BH, H and SM), respectively (p < 0.05). The positive effects of the bacteria application also extends for the stress indicators including: cortisol (0.175, 0.3 and 0.335 µM/ml), glucose (0.9, 1.25 and 0.6 µg/ml) and lactate (2.7, 3 and 3.35) µg/ml and plasma electrolytes consisting of Na(+) (178.5, 175.43 and 175.8 mmol/l) and Cl(-)(123.85, 119.30 and 118.43 mmol/l) in all sampling stages (BH, H and SM), respectively (p < 0.05). Put it all together, P. acidilactici, acting as a probiotic, helps reducing stress symptoms in green terror fish.