Use of <i>Pergularia tomentosa</i>Plant Enzymatic Coagulant System in Fresh Cheese-Making

Introduction: Pergularia tomentosa is a saharan spontaneous plant belonging to Asclepiadacea family. It has several medicinal properties and uses in the human food as an auxiliary in cheese-making. The purpose of this study is the extraction of the coagulant enzymatic system (C.E.S) from the plant dried leaves and its use in the preparation of fresh cheese. Materials & Methods: The determination of the coagulant activity was carried out on a skimmed milk powder by Berridge’s method. Cow’s milk used for coagulant strength determination and cheese-making. Proteolytic activity. The chemical determinations of raw milk were carried out according to AFNOR analysis methods. A descriptive sensory evaluation was performed to determine the organoleptic properties of the produced cheese. The proteolytic activity was determined by measuring the aspartyl-protease units. Results: The main results of the physico-chemical characterization show that its coagulant activity (R.U mL-1) was 97.19 ± 4.67, coagulant strength (SU) was 230.05 ± 0.37, proteolytic activity (μg·mL-1) was 0.33 ± 0.04 and coagulant activity/proteolytic activity ratio was 294.51. For the cheese made, it has presented the following results as % (g/100g): titrable acidity 0.80 ± 0.005, total dry extract 30.48 ± 1.490, fat 2.32 ± 0.02, fresh yield 11.5 ± 0.09, dry yield 30.06 ± 0.05. Moreover, sensory analysis results showed that fresh cheese prepared from Pergularia tomentosa dried leaves diluted enzymatic extract filtrate was characterized by whitish color, smooth texture, with acceptable and satisfying lactic taste. Conclusion: The plant enzymatic system studied could be added to the list of plant rennet substitutes and, thus, contributes to the manufacture of enzymatic coagulation cheese.

A Study of the Enzymatic Hydrolysis of Fish Frames Using Model Systems

A model system was employed to study the operating conditions and primary parameters of enzymic hydrolysis of cod proteins. Pancreatin, papain, and bromelain were used to hydrolyse minced cod fillets under controlled conditions and with the rate of hydrolysis being continually monitored via both the pH-stat and TNBS method. The two methods were compared and evaluated. The rate of protein solubilisation was plotted against the degree of hydrolysis (DH). Dry fish protein hydrolysate (FPH) powders having short, medium and high degrees of hydrolysis (DH of approximately 8%, 11% and 16% respectively) were produced and analysed for their molecular weight distribution, using size exclusion chromatography. Almost complete protein solubilisation (75 g soluble protein per kg hydrolysis solution) could be achieved within an hour, at 40oC, at 1% enzyme/substrate ratio (w/w) with papain and bromelain. The pH-stat was found capable of continuously following the rate of hydrolysis but only at low DH. The TNBS could be accurately used even at high DH to estimate the percentage of the peptide bonds cleaved, but required chemical analysis of withdrawn samples.

Enzymatic hydrolysis of silkworm pupa and its allergenicity evaluation by animal model with different immunization routes

Silkworm pupa is a nourishing food with high nutritional value,but its consumption has been greatly limited given its allergenicity.Enzyme hydrolytic technique is recognized as an effective method to reduce the allergenicity of protein.In this study,we aimed to investigate the effect of enzymolysis on the allergenicity of silkworm pupa.Crude silkworm pupa protein was extracted through alkali extraction and acid precipitation,which included 5 proteins with the molecular weights ranging from 34 kDa to 76 kDa,and silkworm pupa were then hydrolyzed by alkaline protease.The allergenicity of silkworm pupa protein and its enzymatic hydrolysates was evaluated by establishing BALB/c mice model,and the mice were immunized via intragastric gavage and intraperitoneal injection,respectively.The results indicated that the intraperitoneal inj ection immunization route induced more by detecting with antibodies,histamine and Th2-related cytokines.Moreover,mice treated with silkworm pupa protein peptide displayed no obvious allergic symptoms,indicating that enzyme hydrolytic technique could significantly reduce the allergenicity of silkworm pupa.

Unique characteristics of gut microbiota in black snub-nosed monkeys(Rhinopithecus strykeri) reveal an enzymatic mechanism of adaptation to dietary vegetation

DEAR EDITOR,The Myanmar or black snub-nosed monkey(Rhinopithecus strykeri) is a recently discovered and critically endangered colobus primate with an unknown gut microbiota. Here, we characterized and compared the gut microbiota of R. strykeri with those of two closely related snub-nosed monkey species.

Biodegradability of Formulated Enzymatic Solution: An Alternative Waste Reduction System

Solid waste and air pollution are never ending environmental problems that can be partially solved by turning waste into useful bio-products. In this study, enzymatic solutions were developed by formulating enzymatic solutions from fish intestines and fruit peels. This aimed to help in biodegradation of organic solid wastes. Organic waste samples were treated with formulated enzymatic solutions for 7 days in aerated set-up for aerobic degradation. Biodegradability and odor of the waste samples were compared with commercial enzymatic solution. Determination of evolved carbon dioxide (CO2) using gravimetric analysis (GA) was used to determine the percent biodegradability, Dt, of organic solid waste and odor-ranking method was used to determine the odor intensity of the organic waste samples. The result showed that the weight loss and percentage biodegradability of organic waste sample treated with formulated enzymatic solution is comparable with that of the commercial enzymatic solution. In terms of odor intensity, waste treated with the product, formulated enzymatic solution (FES) is also comparable to the waste treated with commercial enzymatic solution. Thus, implying that the developed FES is as efficient as that of the commercial enzymatic solution. These significant results will be helpful to future researchers in providing ways on improving the degradation of organic solid waste and mitigating the increasing glitches on our environment. Enzyme kinetics, physical and chemical properties studies of the solution were recommended for future researchers to accurately determine the effectiveness of the formulated enzymatic solution in the degradation of organic solid waste.

Enzymatic Hydrolysis of Fish Frames Using Pilot Plant Scale Systems

Papain was used to hydrolyse fish frames under controlled conditions at a batch-pilot plant scale- process, for the production of fish protein hydrolysates (FPH). Mass balance calculations were carried out so that the rate of hydrolysis, rate of protein solubilisation and yields could be estimated. Almost complete hydrolysis could be achieved in 1 hour, at 40oC, with no pH adjustment, at 0.5% (5 g.kg-1) enzyme to substrate ratio (E/S, were S is Kjeldahl protein) using whole fish frames (including heads and flaps). This was achieved both with the addition of water (1/1 to 2/1 frames/water) but more importantly from commercial considerations without the initial addition of water (after mincing of the fish material). The degree of protein solubilisation ranged between 71% - 86% w/w. Four different processes are described, namely: 1) a soluble spray-dried FPH powder 2) a liquid FPH 3) a partly soluble, spray dried FPH powder and 4) a crude, drum-dried protein for animal consumption. The amino acid profile of the FPH was identical to that of the parent substrate (fish frames).

Enzymatic debridement shall not modify the global strategy for mass burn events

We read with interest the letter by Surowiecka et al.[1]about early burn wound excision in mass casualty events.We couldn’t agree more with their statement about the benefit of early burn wound excision.Still,we doubt whether applying this strategy to every patient during a mass burn event could be realistic.Of note,while there is an undisputed consensus that early burn wound excision is the gold standard of burn care,what‘early’actually means is still debated.Depending on the authors,the corresponding time limit typically varies from 24 h to a few days[2,3].

Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Enzymatic preparation of mono-and diacylglycerols:A review

Monoacylglycerols(MAGs) and diacylglycerols(DAGs) are partial glycerides widely used in food industry. They are safe and non-toxic food emulsifiers, especially for MAGs. MAGs account for approximately 75% of the total emulsifiers in food industry worldwide. DAGs are recognized as functional cooking oils, they can suppress body fat accumulation and postprandial serum triacylglycerols(TAGs) level. The traditional production of MAGs and DAGs is based on the chemical method, which requires high reaction temperature usually up to 200–260 ℃. Such high temperature is not suitable for oil containing heat sensitive polyunsaturated fatty acids. Enzymatic approach has been received increasing attentions. Enzymatic production of partial glycerides to replace chemical processes has been in industry, particularly for DAGs production as the products have been claimed as a functional and nutritional oil. Enzyme technology for the processing of oils and fats has been moved to industry step by step and case by case during the last 20 years. More and more applications are particularly moving into bulky oils and fats processing. At the same time, the cost of enzymes as a commercial product is reducing steadily. This review summarized the recent 15 years advances on the the enzymatic preparation of MAGs and DAGs. The critical process parameters under different reaction routes were presented and emphasized. The reaction media not only increased the homogeneity of the reaction system, but also shifted the reaction equilibrium towards the target product generation, and this part was stated in detail. In addition, the patent evaluation was included, and the application of MAGs and DAGs was covered.

Semi-Automated Enzymatic Determination of Ethanol in Beverages: Collaborative Study for RIDA®CUBE Ethanol

Easy and quick methods to quantify ethanol reliably in beverages are always important. In 2022, the EnzytecTM Liquid Ethanol test kit was approved as AOAC Official MethodSM 2017.07 Final Action after a collaborative study was conducted with different beverages such as kombucha, juices, and beer. During set-up of this collaborative test, small sized companies asked to include the RIDA®CUBE Ethanol/RIDA®CUBE SCAN device since it is easy to use, suitable for a few samples only and contains the identical reagents as the EnzytecTM Liquid system. It is applicable to quantify ethanol in diluted kombucha, fruit juices, and alcohol-free beer samples around 0.5% alcohol-by-volume within 12 min. The overall relative reproducibility standard deviation across a wide concentration range for kombucha, was calculated to be 6.29%. Analysis of juices and beer showed an overall higher variation with an estimated overall RSD(R) value by regression of 14.4%. The data obtained by this collaborative study show that the RIDA®CUBE Ethanol in combination with the RIDA®CUBE SCAN device is suitable to quantify ethanol from matrices representing important alcohol-free liquid food categories.

Polyphenols extracted from Aronia melanocarpa by combined enzymatic and ultrasonication and their antioxidant potential and antimicrobial activity

The extraction of polyphenols from Aronia melanocarpa was carried out using a combination of enzymatic and ultrasound.After single-factor and orthogonal design and experiment,the optimized polyphenol extraction conditions were 1%enzyme,1:40 material-to-liquid ratio,55℃,60 min ultrasonication,70%ethanol,and the final extraction amount was 88.634 mg/g,which displayed a 25.15%and 34.08%improvement compared with the single ultrasonication and enzymatic extraction methods,respectively.Significant antibacterial effects of polyphenols were shown against Staphylococcus aureus,Escherichia coli and Bacillus subtilis.Further antioxidation effects were evaluated,and the superoxide anion radical scavenging rate,hydroxyl radical scavenging rate and DPPH free radical scavenging rate reached 45.2%,83.5%and 85.4%,respectively.This combined enzymatic and ultrasonic extraction method exhibited the advantages of high extraction rate,saving solvent consumption and extraction time,but also provided a new method for the development and utilization of natural antimicrobial and antioxidant health products.

New possibility for PET plastic recycling by a tailored hydrolytic enzyme

Plastic waste puts a huge burden on the ecosystem due to the current lack of mature recycling technology.Poly(ethylene terephthalate)(PET)is one of the most produced plastics in the world.Enzymatic decomposition holds the promise of recovering monomers from PET plastic,and the monomers can be used to regenerate new PET products.However,there are still limitations in the activity and thermal stability of the existing PET hydrolases.The recent study by Lu et al.introduced a novel PET hydrolase via machine learning-aided engineering.The obtained PET hydrolase showed excellent activity and thermal stability in the hydrolysis of PET and is capable of directly degrading large amounts of postconsumer PET products.This approach provides an effective method for recycling PET waste and is expected to improve the current state of plastic pollution worldwide.

Irrigation regimes modulate non-structural carbohydrate remobilization and improve grain filling in rice(Oryza sativa L.)by regulating starch metabolism

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets. Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling. Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered(WW) and alternate wetting and moderate soil drying(AWMD). Compared with the WW treatment,the activities of ADP glucose pyrophosphorylase(AGPase), starch synthase(StSase) and starch branching enzyme(SBE), and the accumulation of non-structural carbohydrates(NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment. After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase(SPS) and sucrose synthase in the synthetic direction(SSs)were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD. During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction(SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.However, there were no significant differences in vascular microstructures. The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime. We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

Characterization of a Novel Esterase Belonging to Family V from Marinobacter flavimaris

Lipolytic enzymes have attracted enormous attentions because of their ability in ester hydrolysis,ester synthesis,transesterification and other biochemical reactions.Bacteria are important sources of lipolytic enzymes applied in industry.Here,a novel lipolytic enzyme encoded by esterase gene est1347 was identified in Marinobacter flavimaris WLL162,and was purified and characterized.The lipolytic enzyme Est1347 consisted of 312 amino acid residues and a 21-amino-acids N-terminal signal peptide with a predicted molecular weight of 34.2 kDa.It belongs to family V of bacterial lipolytic enzymes based on the amino acid sequence homology analysis.Est1347 is a mesophilic and alkali-resistant enzyme with the highest activity at 45℃and pH 8.5;it is stable at temperatures below 50℃and pH 7.5–11.0.Est1347 showed a preference for middle-length chain substrate p-NPC10 and a wide range of other substrates.The Km,Vmax,Kcat and Kcat/Km values of Est1347 for p-NPC10 in pH 8.5 at 45℃were 0.9411 mmol L^(−1),1285μmol min^(−1)mg^(−1),698.91 s^(−1)and 743.65 s^(−1)(mmol L^(−1))^(−1),respectively.It is also tolerant to the metal ions,organic solvents and detergents.In conclusion,the esterase Est1347 laid a foundation for further study of bacterial lipolytic enzyme family V.

Chitosan Nanoparticles as Biostimulant in Lettuce (Lactuca sativa L.) Plants

Biodegradable nanoparticles such as chitosan nanoparticles (CSNPs) are used in sustainable agriculture since theyavoid damage to the environment;CSNPs have positive effects such as the accumulation of bioactive compoundsand increased productivity in plants. This study aimed to investigate the impact of applying CSNPs on lettuce,specifically focusing on enzymatic activity, bioactive compounds, and yield. The trial was conducted using a completelyrandomized design, incorporating CSNPs: 0, 0.05, 0.1, 0.2, 0.4, and 0.8 mg mL−1. The doses of 0.4 mg mL−1improve yields up to 24.6% increases and 0.1 mg mL−1 of CSNPs increases total phenols by 31.2% and antioxidantcapacity by 34.6%. In addition, when low concentrations of CSNPs (0.05 and 0.1 mg L−1) were applied, anincrease in catalase was determined. The CSNPs represent a good alternative to be used as a biostimulant in sustainableagriculture because they improve the yield and quality of lettuce by increasing the bioactive compounds.

Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb

This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd and/or Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd and/or Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction- denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.

Enzymatic vitrectomy for diabetic retinopathy and diabetic macular edema

The aim of this paper is to determine the role of enzymatic vitrectomy performed by intravitreal injection of autologous plasmin enzyme(APE)in the management of diabetic retinopathy and diabetic macular edema(DME).Diabetic patients with proliferative diabetic retinopathy or DME and evident posterior hyaloid adherence to the retinal surface were included.All cases were treated with an initial intravitreal injection of APE and reevaluated one month later,measuring changes in best-corrected visual acuity(BCVA),macular thickness and the status of the posterior hyaloid.A second APE injection was performed in cases with no evident posterior vitreous detachment(PVD)after the initial treatment.Sixty-three eyes were included in the present review.A complete PVD appeared in 38%of cases(24 eyes)after one injection of plasmin and the total increased to 51%(32 eyes)after the second injection,separated at least by one month.The central macular thickness improved in all cases(100%)and BCVA in89%.Finally,in 50%of eyes with proliferative diabetic retinopathy,a high reduction of new vessels regression was observed.Enzymatic vitrectomy could be considered a good therapeutic alternative in diabetic retinopathy and macular edema.

Thinning intensity affects microbial functional diversity and enzymatic activities associated with litter decomposition in a Chinese fir plantation

Microbial functional diversity and enzymatic activities are critical to maintaining material circulation during litter decomposition in forests.Thinning,an important and widely used silvicultural treatment,changes the microclimate and promotes forest renewal.However,how thinning affects microbial functional diversity and enzymatic activities during litter decomposition remains poorly understood.We conducted thinning treatments in a Chinese fir plantation in a subtropical region of China with four levels of tree stem removal（0,30,50,and 70%）,each with three replicates,and the effects of thinning on microbial functional diversity and enzymatic activities were studied 7 years after treatment by collecting litter samples four times over a 1-year period.Microbial functional diversity and enzymatic activities were analyzed using Biolog Ecoplates（Biolog Inc.,Hayward,CA,USA）based on the utilization of 31 carbon substrates.Total microbial abundance during litter decomposition was lower after the thinning treatments than without thinning.Microbial functional diversity did not differ significantly during litter decomposition,but the types of microbial carbon-source utilization did differ significantly with the thinning treatments.Microbial cellulase and invertase activities during litter decomposition were significantly higher under the thinning treatments due to changes in the litter carbon concentration and the ratios of carbon and lignin to nitrogen.The present study demonstrated the important influence of thinning on microbial activities during litter decomposition.Moderate-intensity thinning may maximize vegetation diversity and,in turn,increase the available substrate sources for microbial organisms in litter and promote nutrient cycling in forest ecosystems.

Cloning,expression in Escherichia coli,and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology （more than 60%） with bacterial laccases in the genome and protein databases and the highest degree of similarity （61% identity） was observed with the multicopper oxidase of KlebsieUa sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS （2,2＇-azino-bis（3-ethylbenzthiazolinesulfonic acid） and DMP （2,6-dimethoxyphenol）, respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Film-mulched continuous ridge-furrow planting improves soil temperature,nutrient content and enzymatic activity in a winter oilseed rape field,Northwest China

Film mulching system is a widely employed agricultural practice worldwide. However, the effects of different planting and mulching patterns on soil nutrient content and enzymatic activity have not been well documented. In this study, we examined the impact of four planting and mulching patterns（including control, flat planting without mulching; M1, flat planting with film mulching; M2, ridge-furrow planting with film mulching on both ridges and furrows; and M3, ridge-furrow planting with film mulching on continuous ridges） on the seed yield of winter oilseed rape, soil moisture, soil temperature, soil organic carbon（SOC） content, soil nutrient content, and soil enzymatic activity over three growing seasons from 2012 to 2015 in a winter oilseed rape field in the semi-arid area of Northwest China. Seed yield of winter oilseed rape, soil moisture, soil temperature, enzymatic activities, and contents of nitrate-nitrogen, available phosphorus, and available potassium were all significantly higher in mulching treatments（M1, M2 and M3） than in control treatment over the three growing seasons, whereas SOC content was significantly lower in mulching treatments than in control treatment during 2013–2014 and 2014–2015. Among the three mulching treatments（M1, M2 and M3）, the M3 treatment showed consistently higher seed yield, SOC content, nutrient contents, and enzymatic activities than the other two treatments. Seed yield of winter oilseed rape was 41.1% and 15.0% higher in M3 than in M1 and M2, respectively. SOC content and soil enzymatic activities in the top 0–20 cm soil layers and nitrate-nitrogen content in the top 0–30 cm soil layers were all significantly higher in M3 than in M1 and M2. Therefore, we advise the ridge-furrow planting with film mulching on continuous ridges（i.e., M3） as an efficient planting and mulching pattern for sustainably improving the seed yield of winter oilseed rape and preserving soil fertility in the semi-arid area of Northwest China.