Enzymatic Treatment Research Articles

Improving functional merit of commercially available pea protein isolate by employing a combination of physical, chemical, and enzymatic modification

Pea protein-based diets have garnered global attention as a viable alternative to meat or primary protein sources. Nevertheless, the utilization of pea protein is hindered by issues such as solubility, and emulsifying properties. This research presents a combined set of techniques employed to commercially available Pea Protein Isolate (PPI) to effectively address major setbacks associated with the usage of pea protein in diverse industrial sectors, especially meat. The set integrates pH at 10.0, sonication at three different magnitudes (1,2 and 4 W/mL), heat at 60 °C, and enzyme hydrolysis through Flavourzyme at three different enzyme-to-substrate ratios (0.25%, 0.5%, and 1.0%). Among the twelve treatments, PPI11, PPI12, and PPI13 showed promising outcomes for artificial meat. The treatments exhibited favorable solubility, gelling, emulsifying, and foaming properties while containing the optimum and desirable protein size after undergoing partial hydrolysis comprehended in the gel electrophoresis results. Solubility (for PPI11, PPI12, PPI13) had seen significant (p < 0.05) improvement from 31.03% ± 2.11% (Control) to 64.43% ± 2.09%, 66.23% ± 2.11%, and 69.87% ± 1.57%, accompanied by changes in protein morphology in scanning electron microscopy. Furthermore, treated PPIs were applied to stabilize sunflower oil-in-water emulsion, showing a significant escalation in emulsion capacity (38.46%, 39.42% and 66.61% increase for PPI11, PPI12, PPI13), foaming capacity (46.88%, 51.20%, and 69.60% increase for PPI11, PPI12, PPI13), and foaming stability (30 min) (23.97%, 28.70%, and 30.22% increase for PPI11, PPI12, PPI13). Gelling properties were decisive in this study (2.512 ± 0.1 N, 2.604 ± 0.1 N, and 2.168 ± 0.3 N, for PPI11, PPI12, and PPI13), predominantly in plant-based meat analog perspective. So the mechanism proposed through this research will enable pea protein treated with this set of operations, combining physical, chemical and enzymatic treatments, to further its journey towards considerably improved meat analogs.

Comparative studies on enzymatic efficacy and microbiological properties of Garbage Enzyme produced from various fruits and vegetables peels in biodegradation of cow dung

The present work was conducted to study the enzymatic potentials and microbiological properties of Garbage Enzyme produced from various fruits and vegetables peels. Completely randomized designed experiment was framed with seven treatments each with fruits and vegetable peels separately. All the treatments exhibited positive result for Amylase Test, Lipase Test, Cellulase Test, Urease Test, Protease Test, MR Test and Alcohol and low pH, indicating that Garbage enzyme produced from fruit and vegetable wastes contained mixture of enzymes, alcohol and acid brought about by microbial fermentation. Highest enzymatic efficacy was shown by T3- Pomegranate followed by T6- Banana. GE produced by vegetable wastes was less effective than fruit peels and maximum enzymatic activity exhibited by T1-Radish. A total of 27 amylase producing bacteria, 27 protease producing bacteria, 26 lipase producing bacteria, 26 cellulase producing bacteria 24 urease producing bacteria, 21 pectinase producing bacteria and 01 Amylase producing fungus was screened isolated from all the treatments. Garbage enzyme treatments in tray assay neutralized the alkaline pH of cow dung as garbage enzyme is acidic in nature. Highest Nitrogen (172 Kg/Hec) content was found when treated with garbage enzyme produced with banana peel. It is concluded that Garbage enzyme contains various enzymes, acid, and alcohol, can be utilized as a low cost alternative to biodegrade wastes to reduce pollution load of our environment. Keywords: Garbage Enzyme, vegetable and fruit wastes, enzyme activity, biodegradation

Characterization, Antioxidant Capacity and Protective Effect of Peptides from Cordyceps militaris Cultivated with Tussah Pupa on Oxidative Injured HepG2 Cells.

The antioxidant capacity and protective effect of peptides from protein hydrolysate of Cordyceps militaris cultivated with tussah pupa (ECPs) on H2O2-injured HepG2 cells were studied. Results indicated ECP1 (<3 kDa) presented the strongest antioxidant activity compared with other molecular weight peptides. Pretreated with ECPs observably enhanced survival rates and reduced apoptosis rates of HepG2 cells. ECPs treatment decreased the ROS level, MDA content and increased CAT and GSH-Px activities of HepG2 cells. Besides, the morphologies of natural peptides from C. militaris cultivated with tussah pupa (NCP1) and ECP1 were observed by scanning electron microscopy (SEM). Characterization results suggested the structure of NCP1 was changed by enzymatic hydrolysis treatment. Most of hydrophobic and acidic amino acids contents (ACC) in ECP1 were also observably improved by enzymatic hydrolysis. In conclusion, low molecular weight peptides had potential value in the development of cosmetics and health food.

Bacterial cellulose nanocrystals obtained through enzymatic and acidic routes: A comparative study of their main properties and in vitro biological responses

Cellulose nanocrystals (CNCs) are crystalline domains isolated from cellulosic fibers. They have been utilized in a wide range of applications, such as reinforcing fillers, antibacterial agents and manufacturing of biosensors. Whitin this context, the aim of this work was to obtain and analyze CNCs extracted from bacterial nanocellulose (BNC) using two distinct methods combined with milling pre-treatment: an acidic hydrolysis using 64 % sulfuric acid and an enzymatic hydrolysis using a commercial cellulase enzyme mixture. The CNCs obtained from the enzymatic route (e-CNCs) were observed to be spherical nanoparticles with diameter of 56 ± 11 nm. In contrast, the CNCs from the acid hydrolysis (a-CNCs) appeared as needle-shaped nanoparticles with a high aspect ratio with lengths/widths of 158 ± 64 nm/11 ± 2 nm. The surface zeta potential (ZP) of the a-CNCs was −30,8 mV, whereas the e-CNCs has a potential of +2.70 ± 3.32 mV, indicating that a-CNCs consisted of negatively charged particles with higher stability in solution. Although the acidic route resulted in nanocrystals with a slightly higher crystallinity index compared to the enzymatic route, e-CNCs was found to be more thermally stable than BNC and a-CNCs. Here, we also confirmed the safety of a-CNCs and e-CNCs using L929 cell line. Lastly, this article describes two different CNCs synthesis approaches that leads to the formation of nanoparticles with different dimensions, morphology and unique physicochemical properties. To the best of our knowledge, this is the first study to yield spherical nanoparticles as a result of BNC enzymatic treatment.

The Combination of Enzymes and Conidia of Entomopathogenic Fungi against Aphis gossypii Nymphs and Spodoptera frugiperda Larvae.

The slow action of fungi is one of the biggest challenges in using entomopathogenic fungi. A promising alternative to reduce the time of action is to combine conidia with extracellular enzymes. This study aimed to characterize the production of Pr1 subtilisin protease and lipases by Beauveria bassiana and Metarhizium anisopliae in different culture media and to evaluate the efficiency of the enzymatic treatment against Aphis gossypii and Spodoptera frugiperda. The isolates were cultivated in five different liquid cultures, and, after 7 days, the culture was filtered and centrifuged, and the activity of the Pr1 and lipases was measured. The fungi cultured in a Luria-Bertani broth medium had the highest activity of proteases and lipases. The mortality of A. gossypii nymphs treated with conidia 7 days after the treatment was 39% (JEF-410), 76.5% (JEF-492), 74.8% (ERL-836), and 70.9% (JEF-214). The B. bassiana JEF-410 supernatant combined with conidia increased the fungal virulence at day 5 and day 6 after treatment. When S. frugiperda larvae were treated with B. bassiana JEF-492 conidia combined with its supernatant, the time of infection was shorter compared to the larvae treated with conidia only. Once the supernatant was incubated at 37 °C, the relative activity decreased from 100% to 80% after 2 h and to 45% after 24 h. The results suggest that the supernatant of entomopathogenic fungi may be formulated and used as a biopesticide in an efficient strategy for the biological control of pests.

The potential of glucosidase and glucose oxidase for aroma improvement in concentrated peach puree based on volatilomics and metabolomics

Cooked off-flavor was produced during the processing of concentrated peach puree (CPP), which led to aroma deterioration. Enzymatic treatment was beneficial in eliminating off-flavors and improving the aroma quality. Herein, the efficacy of glycosidase (AR2000), glucose oxidation (GOD), and their combination on the inhibition of off-flavors and aroma enhancement were evaluated. Compared with CPP, contents of benzaldehyde, benzyl alcohol, nonanal, and linalool increased by 198%, 1222%, 781%, and 71% after AR2000 treatment via the metabolisms of shikimate, glucose, linoleic acid, and linolenic acid, leading to the strengthening of floral and grassy. Due to the removal of 1-octen-3-one via linolenic acid metabolism, cooked off-flavor could be significantly weakened by GOD. Furthermore, Furthermore, the combination of AR2000 and GOD could not only inhibit the production of 1-octen-3-one to weaken the cooked note but also enhance grassy and floral attributes via the increase of aldehydes and alcohols.

Effect of ultrasound-assisted enzyme pretreatment on γ-aminobutyric acid content, eating quality and starch properties of germinated brown rice

A novel technique has been developed to increase γ-aminobutyric acid levels and enhance the quality of germinated brown rice (GBR) by ultrasound and enzyme treatment. The combined use of ultrasonic and enzyme treatments notably enhanced the germination rate of brown rice. Additionally, the γ-aminobutyric acid content substantially improved from 18.79 ± 1.87 mg/100 g to 38.60 ± 3.19 mg/100 g when compared to the untreated group. Moreover, the cracks produced on the GBR surface and disruption of the starch structure resulted in increased water absorption, shortening of cooking time, and reduction in GBR hardness, thereby improving the cooking and edible quality of GBR. Additionally, ultrasonic and enzyme treatments increased the amount of fast-digestible starch and enhanced starch digestibility. However, they had minimal impact on the crystal structure, degree of crystallinity, and heat energy changes during the starch pasting process of GBR.

HPV infection and ART: the andrologist’s point of view

Human papilloma virus infection is a common sexually transmitted disease. In addition to its tropism for epithelial mucosae and skin, HPV can also infect the male urogenital tract and strongly bind the sperm head, causing a reduction of semen quality, with detrimental effects on both natural and assisted fertility. An association between HPV and assisted reproductive technology (ART) programs has been reported, and HPV infection of semen seems to have a negative influence on the outcome of these procedures. None of the standard procedures commonly used for sperm selection before ART showed efficacy in eliminating HPV from the sperm head. Several strategies have been suggested to improve viral clearance in semen of infertile patients, such as counseling and anti-HPV vaccination. Moreover, these strategies were able to increase the natural fertility of couples with HPV semen infection. Finally, a modified swim-up with the addition of an enzymatic treatment, despite reducing sperm motility, was reported to eliminate the virus from the sperm head. In this manuscript we propose a flow chart for the management of infertile couples with HPV semen infection. KEY WORDS: HPV, HPV semen infection, assisted reproductive techniques, HPV vaccines.

Optimal probiotic combinations for treating nonalcoholic fatty liver disease: A systematic review and network meta-analysis

BackgroundProbiotic administration is a promising therapy for improving conditions in NAFLD patients. This network meta-analysis aimed to compare and estimate the relative effects of probiotic interventions and identify the optimal probiotic species for the treatment of NAFLD (Nonalcoholic fatty liver disease) patients. MethodsThe PubMed, Web of Science, Embase, and Cochrane databases were searched from inception to 29 January 2024 to identify RCTs that were published in English. The GRADE framework was used to assess the quality of evidence contributing to each network estimate. ResultsA total of 35 RCTs involving 2,212 NAFLD patients were included in the analysis. For primary outcomes, Lactobacillus + Bifidobacterium + Streptococcus exhibited the highest probability of being the finest probiotic combination in terms of enhancing acceptability as well as reducing AST (SMD: -1.95 95% CI: -2.90, -0.99), ALT (SMD = -1.67, 95% CI: -2.48, -0.85), and GGT levels (SMD = -2.17, 95% CI: -3.27, -1.06). In terms of the secondary outcomes, Lactobacillus + Bifidobacterium + Streptococcus was also the best probiotic combination for reducing BMI (SMD = -0.45, 95% CI: -0.86, -0.04), LDL levels (SMD = -0.45, 95% CI: -0.87, -0.02), TC levels (SMD = -1.09, 95% CI: -1.89, -0.29), and TNF-α levels (SMD = -1.73, 95% CI: -2.72, -0.74). ConclusionThis network meta-analysis revealed that Lactobacillus + Bifidobacterium + Streptococcus may be the most effective probiotic combination for the treatment of liver enzymes, lipid profiles, and inflammation factors. These findings can be used to guide the development of a probiotics-based treatment guideline for NAFLD since there are few direct comparisons between different therapies.

Industrial Distillation Fractions of Garlic Essential Oil, Design, Synthesis, and Antifungal Activity Evaluation of Aliphatic Substituted Trisulfide Derivatives.

Garlic oil has a wide range of biological activities, and its broad-spectrum activity against phytopathogenic fungi still has the potential to be explored. In this study, enzymatic treatment of garlic resulted in an increase of approximately 50 % in the yield of essential oil, a feasible GC-MS analytical program for garlic oil was provided. Vacuum fractionation of the volatile oil and determination of its inhibitory activity against 10 fungi demonstrated that garlic oil has good antifungal activity. The antifungal activity levels were ranked as diallyl trisulfide (S-3)>diallyl disulfide (S-2)>diallyl monosulfide (S-1), with an EC50 value of S-3 against Botrytis cinerea reached 8.16 mg/L. Following the structural modification of compound S-3, a series of derivatives, including compounds S-4~7, were synthesized and screened for their antifungal activity. The findings unequivocally demonstrated that the compound dimethyl trisulfide (S-4) exhibited exceptional antifungal activity. The EC50 of S-4 against Sclerotinia sclerotiorum reached 6.83 mg/L. SEM, In vivo experiments, and changes in mycelial nucleic acids, soluble proteins and soluble sugar leakage further confirmed its antifungal activity. The study indicated that the trisulfide bond structure was the key to good antifungal activity, which can be developed into a new type of green plant-derived fungicide for plant protection.

Quality improvement of banana fiber through sequential enzymatic treatment

Quality improvement of banana fiber through sequential enzymatic treatment

In vitro digestibility and solubility of phosphorus of three plant-based meat analogues.

Interest in plant-based meat analogues has increased and can be expected to be applied to pet foods, which necessitates the understanding of the nutrient supply in those foods. Our primary aim was to advance our understanding of the digestive properties of sterilized plant-based meat analogues. The impact of the preparatory processing steps on the solubility of meat analogues was studied. Meat analogues were made by mixing water, salt, and wheat gluten with soy protein isolate, pea protein isolate, or faba bean concentrate. Mixed materials were processed into model meat analogues using shear cell technology. Products were canned in water or gravy and sterilized. An animal-based canned pet food was made as a reference. Products sampled at the processing steps (mixing, shearing, sterilization) were digested in vitro. Samples of digestate were taken at the gastric phase (0 and 120 min) and small intestinal phase (120, 200, 280, and 360 min) for analysis of protein hydrolysis. The extent digestion of nitrogen and dry matter was determined at the end of incubation. Total phosphorus, soluble phosphorus after acid treatment, and after acid and enzymatic treatment were determined. The degree of hydrolysis after gastric digestion was low but increased immediately in the small intestinal phase; products based on pea had the highest values (56%). Nitrogen digestibility was above 90% for all materials at each processing step, indicating that bioactive compounds were absent or inactivated in the protein isolates and concentrate. Phytate seemed to play a minor role in meat analogues, but phosphorus solubility was influenced by processing. Shearing decreased soluble phosphorus, but this effect was partly reversed by sterilization. Nutrient digestibility as well as phosphorus solubility in plant-based products was higher than or comparable with the reference pet food. These findings show that the digestive properties of the tested plant-based meat analogues do not limit the supply of amino acids and phosphorus.

Congenital Sucrase-Isomaltase Deficiency: Same Mutation with Different Clinical Presentations.

Congenital sucrase-isomaltase deficiency is an autosomal recessive inherited disaccharidase deficiency characterized by chronic osmotic diarrhea. In this study, the genotype-phenotype relationships of close relatives of an index case with congenital sucrase-isomaltase deficiency were investigated. A 23-month-old female patient with a sucrase-isomaltase gene c.317G>A (p.C106Y) homozygous mutation was diagnosed as an index case and her pedigree analysis was performed subsequently. The family members with and without sucrase- isomaltase gene mutations were compared in terms of clinical symptoms. The study included 109 cases [mean age ± SD: 22.6 ± 17.2 years (0.1-75 years), 61 males (56%)] of 130 family members of the index case. Sucrase-isomaltase gene c.317G>A (p.C106Y) heterozygous mutation was detected in 27 cases (24.7%); 14 (51.9%) were male and had a mean age of 23.2 ± 18.3 years. The most common complaints of 12 (44.4%) symptomatic patients with mutations were abdominal pain (37%), gas irritability (33.3%), bloating (22.2%), and foul-smelling stools (18.5%). Compared with the cases without mutation, a statistically significant difference was observed in the incidence of gas irritability, foul-smelling stool, ≥2 gastrointestinal symptoms, postprandial complaints, and food allergy (P = .005, P = .047, P = .049, P = .017, P = .021, respectively). Sacrosidase enzyme replacement was applied to 7 patients whose symptoms did not improve with dietary elimination. Clinical response was obtained after enzyme treatment. Despite its autosomal recessive inheritance, congenital sucrase-isomaltase deficiency can also be symptomatic in heterozygous individuals. Further studies are required to clarify the genotype-phenotype relationship and management of the disease.

(369) - A-Antigen Re-Expression and Prevention of Hyperacute Rejection Following In Vivo Enzymatic Treatment in a Model of ABO-Incompatible Transplantation

(369) - A-Antigen Re-Expression and Prevention of Hyperacute Rejection Following In Vivo Enzymatic Treatment in a Model of ABO-Incompatible Transplantation

Optimization of microalgae protein extraction from Scenedesmus obliquus and investigating its functional properties

The objective of this work was to evaluate alkaline treatment with enzymatic approaches for the optimal extraction of proteins from Scenedesmus obliquus microalgae. The experiments were designed with an emphasis on the alkaline treatment period and the enzyme-to-substrate ratio utilizing response surface methodology (RSM) with central composite design (CCD). Higher enzyme concentrations led to better extraction rates and efficiency, according to the findings. Cellulase demonstrated extraction efficiencies between 16.88% and 21.42%, but the multi-enzyme complex had efficiencies ranging from 15.28% to 27.87%. With an extraction rate of 19.13%, alkaline treatment was less advantageous than enzymatic treatment due to its longer procedure. Microalgal proteins' functional characteristics were also looked at. These proteins demonstrated foaming capacity, oil absorption, and emulsification ability, making them suitable alternatives to animal proteins in food systems such as egg white and soybean. Although microalgal protein powder had lower foam capacity (20%), stability (25%), fat absorption capacity (124%), and emulsification ability compared to egg white powder, it still exhibited favorable functional properties, highlighting its potential for various food applications. Further enhancement of the functional properties can be achieved by optimizing the environmental conditions during extraction.

Detection of Microplastics and Heavy Metals in Famous Edible Molluscs in Can Gio Biosphere Reserve of Vietnam

Can Gio, designated in 2000, is the first Biosphere Reserve of Vietnam. In recent years, local agriculture, tourism, households, nearby industrial zones, and maritime transport have released substances into rivers flowing through Can Gio Mangrove to the East Sea and transported into creatures. This study aims to investigate microplastics and heavy metals in the famous seafood of Can Gio. A Raman microscope was used to identify microplastics from the tissue of oysters, grease snails, and clams after enzymatic treatment with trypsin. Heavy metals were quantified with ICP-MS after acidic treatment. As a result, microplastics were found in almost all samples, even up to 10 in a random individual of bivalve. Fibers dominated, especially from 30 to 150 µm long. The metal with the highest content was zinc, followed by copper, especially in oysters. Non-essential metals were also present in the soft tissue in the order As &gt; Pb &gt; Cd. The accumulation of metals in Can Gio’s molluscs was generally lower than in other regions and still in the allowed range for consumption of Vietnamese Standard. Although there have not been any Vietnamese regulations on the permissible level of microplastics for consumption, regular seafood intake will increase the risk of chronic poisoning and unknown disorders caused by the accumulation of microplastics and heavy metals.

Valorisation of low-density polythene (LDPE) waste into fuels and chemicals through alkane hydroxylase and microbial treatment

Polythene exhibits low reactivity and slow biodegradability due to its highly reduced hydrocarbon structure. In this study, a process has been developed for the transformation of polythene waste into fuel and its precursor molecule by microbial and enzymatic treatments. In first step polythene was pretreated with Paenibacillus alvei. Through fermentation process for 15 days and products released after fermentation were subsequently bio-transformed into alcohols and acids corresponding to its hydrocarbons, employing Alkane hydroxylase (AlkB). On fermentation using Paenibacillus alvei, 16.8% weight loss was observed after 15 days and surface modification was analyzed through AFM and SEM. The major products released during microbial pretreatment analyzed through Chromatography–High-Resolution Mass Spectrometry (GC-HRMS) were pentane, hexane, heptadecane, dodecane, tetracontane, bromotetradecane, methyl esters, hexadecane, etc. which were converted in second step into hexadecanol, octadecanol, octadecanoic acid, and heptadecanoic acid, etc. with the help of Alkane hydroxylase on enzymatic transformation. These findings suggested that the microbial pretreatment results in a release of alkanes, some fatty acids, and esters using Paenibacillus alvei Among these, AlkB facilitated the transformation of C16 and C15 hydrocarbons into hexadecanol, and pentadecanol respectively. This process holds the potential as an alternative to traditional fuels, and an optimization process can be pursued to enhance the overall yield.

Proteus mirabilis biofilm expansion microscopy yields over 4-fold magnification for super-resolution of biofilm structure and subcellular DNA organization

Bacterial biofilms form when bacteria attach to surfaces and generate an extracellular matrix that embeds and stabilizes a growing community. Detailed visualization and quantitative analysis of biofilm architecture by optical microscopy are limited by the law of diffraction. Expansion Microscopy (ExM) is a novel Super-Resolution technique where specimens are physically enlarged by a factor of ∼4, prior to observation by conventional fluorescence microscopy. ExM requires homogenization of rigid constituents of biological components by enzymatic digestion. We developed an ExM approach capable of expanding 48-h old Proteus mirabilis biofilms 4.3-fold (termed PmbExM), close to the theoretic maximum expansion factor without gross shape distortions. Our protocol, based on lytic and glycoside-hydrolase enzymatic treatments, degrades rigid components in bacteria and extracellular matrix. Our results prove PmbExM to be a versatile and easy-to-use Super-Resolution approach for enabling studies of P. mirabilis biofilm architecture, assembly, and even intracellular features, such as DNA organization.

Bioplastic films from cassava peels: Enzymatic transformation and film properties

Annually, millions of tons of cassava peels are generated as agro-waste. This research repurposed entire cassava peels into biodegradable films as a sustainable alternative to single-use plastics. Enzymes (cellulase, xylanase, and glucanase) were employed to catalyze the process, and cassava peels were effectively converted into films, which exhibited comparable or even better mechanical properties and water resistance to commercial packaging materials. The films also presented high water solubility of 44–50% and good light barrier properties, with 15–23% transmittance values for visible light and less than 3% for UV light. Extended enzymatic treatment time resulted in films characterized by increased elasticity, solubility, water resistance, transparency, and smoother surfaces, which can be attributed to the enzymatic breakdown of cassava peel fibers into smaller saccharide units. The optimal enzymatic treatment time was found to be 4 h which produced films with favorable mechanical characteristics, barrier properties, and solubility. A sustainable and ecologically-friendly approach is provided to fully harness cassava peels in fabricating packaging films, particularly suited for food items necessitating dissolution in water.

Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n=12) under different (patho)physiological conditions as well as in human-derived biofluids (n=3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.