Characteristic aroma compounds during the fermentation of Chinese steamed bread fermented with different starters.

The characteristic aroma compounds of Chinese steamed bread (CSB) fermented with different starters were studied using HS-SPME-GC/MS, aroma recombination and omission experiments. The dynamic changes of the microbiota and their function and metabolites during fermentation were analyzed using metagenomics and non-targeted metabolomics. Forty-nine volatile flavor compounds were identified, while 5 characteristic aroma-active compounds were investigated in CSB fermented with commercial dry yeast (AQ-CSB), and 10 were investigated in CSB fermented with traditional starter (NY-CSB). Microbial structure and function analysis showed that Saccharomyces cerevisiae dominated during AQ-CSB fermentation and contributed >95% to its KEGG pathways, while Pediococcus pentosaceus, unclassified Pediococcus, Lactobacillus plantarum, Lactobacillus brevis and unclassified Lactobacillus were predominant in NY-CSB and together had an ~96% contribution to these pathways. NY-CSB showed higher metabolic activity during fermentation, and the characteristic metabolites were mainly involved in carbohydrate, amino acid and lipid metabolism. The characteristic aroma compounds were identified and increased the understanding of the contributions of the microbiota. This may be useful for designing starter cultures that produce CSB with desirable aroma properties.

The complex world of kefir: Structural insights and symbiotic relationships.

Kefir milk, known for its high nutritional value and health benefits, is traditionally produced by fermenting milk with kefir grains. These grains are a complex symbiotic community of lactic acid bacteria, acetic acid bacteria, yeasts, and other microorganisms. However, the intricate coexistence mechanisms within these microbial colonies remain a mystery, posing challenges in predicting their biological and functional traits. This uncertainty often leads to variability in kefir milk's quality and safety. This review delves into the unique structural characteristics of kefir grains, particularly their distinctive hollow structure. We propose hypotheses on their formation, which appears to be influenced by the aggregation behaviors of the community members and their alliances. In kefir milk, a systematic colonization process is driven by metabolite release, orchestrating the spatiotemporal rearrangement of ecological niches. We place special emphasis on the dynamic spatiotemporal changes within the kefir microbial community. Spatially, we observe variations in species morphology and distribution across different locations within the grain structure. Temporally, the review highlights the succession patterns of the microbial community, shedding light on their evolving interactions.Furthermore, we explore the ecological mechanisms underpinning the formation of a stable community composition. The interplay of cooperative and competitive species within these microorganisms ensures a dynamic balance, contributing to the community's richness and stability. In kefir community, competitive species foster diversity and stability, whereas cooperative species bolster mutualistic symbiosis. By deepening our understanding of the behaviors of these complex microbial communities, we can pave the way for future advancements in the development and diversification of starter cultures for food fermentation processes.

Molecular structural modification of myofibrillar protein from oyster (Crassostrea gigas) with oligosaccharides for improving its gel properties.

Glycation is a promising approach to enhance protein gel characteristics in the food industry. The impact of oyster myofibrillar protein (MP) being glycosylated with six oligosaccharides (dextran [Dex]-1 kDa, 5 kDa, 6 kDa, and 10 kDa, xylan [Xyla], and xyloglucan [Xyg]) on structural properties, aggregation behavior and gel properties was investigated in this study. The findings demonstrated that oligosaccharides significantly increased the glycation degree of MP by forming a stable tertiary conformation, increasing the contents of the disulfide bond and hydrogen bonds. Additionally, particle sizes decreased and solubility increased after glycation, improving the gel's strength, water-holding capacity, thermal stability, elastic modulus, and ordered network layout. It was determined that MP-Dex 5 had the best gel properties. The gel strength and water holding capacity of MP-Dex 5 increased by 70.59% and 32.27%, respectively. Molecular dynamics simulations results showed van der Waals energy and electrostatic interactions favor myosin binding to Dex or Xyla units. This study will provide insights into the relationship between molecular structure, aggregation behavior and gel property of oyster MP-oligosaccharide couples, and expand the application of oyster MP in food gels.

6-Shogaol alleviates high-fat diet induced hepatic steatosis through miR-3066-5p/Grem2 pathway.

The purpose of this study is to investigate the mechanism by which 6-shogaol ameliorates hepatic steatosis via miRNA-mRNA interaction analysis. C57BL/6 J mice were fed a high-fat diet (HFD) for 12 weeks, during which 6-shogaol was administered orally. The liver lipid level, liver function and oxidative damage in mice were evaluated. mRNA sequencing, miRNA sequencing, and RT-qPCR were employed to compare the expression profiles between the HFD group and the 6-shogaol-treated group. High-throughput sequencing was used to construct the mRNA and miRNA libraries. Target prediction and integration analysis identified eight potential miRNA-mRNA pairs involved in hepatic steatosis, which were subsequently validated in liver tissues and AML12 cells. The findings revealed that 6-shogaol modulates the miR-3066-5p/Grem2 pathway, thereby improving hepatic steatosis. This study provides new insights into the mechanisms through which 6-shogaol alleviates hepatic steatosis, establishing a foundation for future research on natural active compounds for the treatment of metabolic diseases.

Absorption and intracellular accumulation of food-borne dicarbonyl precursors of advanced glycation end-product in a Caco-2 human cell transwell model.

This study aimed to better understand whether and how the reactive 1,2-dicarbonyl precursors of advanced glycation end products (AGEs), glyoxal (GO) and methylglyoxal (MGO), cross the intestinal barrier by studying their transport in the in vitro Caco-2 transwell system. The results reveal that GO, MGO and Nε-(carboxymethyl)lysine (CML), the latter studied for comparison, are transported across the intestinal cell layer via both active and passive transport and accumulate in the cells, albeit all to a limited extent. Besides, the transport of the dicarbonyl compounds was only partially affected by the presence of amino acids and protein, suggesting that scavenging by a food matrix will not fully prevent their intestinal absorption. Our study provides new insights into the absorption of the two major food-borne dicarbonyl AGE precursors and provides evidence of their potential systemic bioavailability but also of factors limiting their contribution to the overall exposome.

Microfluidic-oriented green synthesis of pepsin-doped gold nanoparticles for colorimetric and photothermal dual-readout detection of food hazards.

Gold nanoparticles (AuNPs)-based immunochromatographic assay has gained popularity as a rapid detection method for food hazards. Synthesizing highly stable AuNPs in a rapid, simple and environmentally friendly manner is a key focus in this field. Here, we present a green microfluidic strategy for the rapid, automated, and size-controllable synthesis of pepsin-doped AuNPs (AuNPs@Pep) by employing glucose-pepsin as a versatile reducing agent and stabilizer. Through combining the colorimetric and photothermal (PoT) properties of AuNPs@Pep, both "signal-off" and "signal-on" formats of microfluidic paper analytical devices (PADs) were developed for detection of a small molecule antibiotic, florfenicol, and an egg allergen, ovalbumin. Compared to the colorimetric mode, a 4-fold and 3-fold improvement in limit of detection was observed in the "signal-off" detection of florfenicol and the "signal-on" detection of ovalbumin, respectively. The results demonstrated the practicality of AuNPs@Pep as a colorimetric/PoT dual-readout probe for immunochromatographic detection of food hazards at different molecular scales.

Integrated ultra-high pressure and salt addition to improve the in vitro digestibility of myofibrillar proteins from scallop mantle (Patinopecten yessoensis).

Myofibrillar protein (MP) is susceptible to the effect of ionic strength and ultra-high pressure (UHP) treatment, respectively. However, the impact of UHP combined with ionic strength on the structure and in vitro digestibility of MP from scallop mantle (Patinopecten yessoensis) is not yet clear. Therefore, it is particularly important to analyze the structural properties and enhance the in vitro digestibility of MP by NaCl and UHP treatment. The findings demonstrated that as ionic strength increased, the α-helix and ß-sheet gradually transformed into ß-turn and random coil. The decrease of endogenous fluorescence intensity indicated the formation of a more stable tertiary structure. Additionally, the exposure of internal sulfhydryl groups increased the amount of total sulfhydryl content, and reactive sulfhydryl groups gradually transformed into disulfide bonds. Moreover, it reduces aggregation through increased solubility, decreased turbidity, particle sizes, and a relatively dense and uniform microstructure. When MP from the scallop mantle was treated with 0.5 mol/L ionic strength and 200 MPa UHP treatment, it had the highest solubility (90.75 ± 0.13%) and the lowest turbidity (0.41 ± 0.03). The scallop mantle MP with NaCl of 0.3 mol/L and UHP treatment had optimal in vitro digestibility (95.14 ± 2.01%). The findings may offer a fresh perspectives for developing functional foods for patients with dyspepsia and a theoretical foundation for the comprehensive utilization of scallop mantle by-products with low concentrations of NaCl.

Degradation Mechanism of Aflatoxin M1 by Recombinant Catalase from Bacillus pumilusE-1-1-1: Food Applications in Milk and Beer.

A bacteria capable of degrading aflatoxin M1 (AFM1) was isolated from African elephant manure. It was identified as Bacillus pumilus by 16s rDNA sequencing and named B. pumilusE-1-1-1. Compared with physical and chemical methods, biological methods have attracted much attention due to their advantages, such as thorough detoxification, high specificity, and environmental friendliness. This work aimed to study the effects of a recombinant catalase (rCAT) from B. pumilusE-1-1-1 on the degradation of AFM1 in pattern solution. The degradation mechanism was further explored and applied to milk and beer. Kinetic Momentum and Virtual Machine Maximum values for rCAT toward AFM1 were 4.1 µg/mL and 2.5 µg/mL/min, respectively. The rCAT-mediated AFM1 degradation product was identified as C15H14O3. Molecular docking simulations suggested that hydrogen and pi bonds played major roles in the steadiness of AFM1-rCAT. In other work, compared with identical density of AFM1, survival rates of Hep-G2 cells incubated with catalase-produced AFM1 degradation products increased by about 3 times. In addition, degradation rates in lager beer and milk were 31.3% and 47.2%, respectively. Therefore, CAT may be a prospective substitute to decrease AFM1 contamination in pattern solution, milk, and beer, thereby minimizing its influence on human health.

Antioxidant potential and protective effect of modified sea cucumber peptides against H2O2-induced oxidative damage in vitro HepG2 cells and in vivo zebrafish model.

In this study, modified sea Cucumber Peptides (SCP) were prepared by reacting with xylooligosaccharide (XOS) and alginate oligosaccharides (AOS) via glycation. Free radical inhibitory and inhibition of oxidative stress of modified SCP was evaluated using human hepatocellular carcinoma (HepG2) cells and zebrafish embryos. LC-MS analysis revealed that SCPs mainly consist of 40 active peptides, with an average molecular weight of 1122.168 Da and an average length of 11 amino acid residues. For amino acid composition, L-Asparagine, L-Methionine, and L-Aspartic Acid were dominant amino acids in SCP. The result showed that the antioxidant ability of SCP against 2,2-Diphenyl-1-picrylhydrazyl (DPPH), superoxide anion radical (O-2), and Hydroxyl Radical (OH) was significantly improved after modification. In HepG2 cells, the modified SCP showed stronger protection than native SCP native against H2O2-induced oxidative stress by enhancing cell viability and reducing radical oxygen species (ROS) generation. The inhibition effect of SCP was increased after modification with XOS and AOS by 13 % and 19 % respectively. Further studies displayed that the activity of antioxidative enzymes, including Superoxide dismutase (SOD), Glutathione Peroxidase (GPx), and catalase (CAT), was remarkably enhanced, whereas malondialdehyde (MDA) level was reduced compared with native SCP and H2O2-treated groups, thus, improving the intracellular antioxidant defenses. The gene expression analysis showed that the mechanism underlying the modified SCP protective effect may be linked with the capability to regulate Nuclear factor-erythroid factor 2-related factor 2 (NRF2) gene expression. The protective effect of modified SCP against H2O2 in vitro was confirmed in vivo by reduced toxicity in zebrafish embryos via improvement of mortality rate, hatching rate, heart beating rate, and deformities of the zebrafish model. However, SCPAOS conjugate displayed greater antioxidant potentials compared to the SCPXOS, the different effects between SCPAOS and SCPXOS could be due to their different antioxidant activity. Thus, modified SCP could be potentially used as a novel nutraceutical in the preparation of anti-aging food and medicine.

Preparation and recognition mechanism study of an scFv targeting chloramphenicol for a hybridization chain reaction-CRISPR/Cas12a amplified fluoroimmunoassay.

Recombinant antibody-based immunoassays have emerged as crucial techniques for detecting antibiotic residues in food samples. Developing a stable recombinant antibody production system and enhancing detection sensitivity are crucial for their biosensing applications. Here, we bioengineered a single-chain fragment variable (scFv) antibody to target chloramphenicol (CAP) using both Bacillus subtilis and HEK 293 systems, with the HEK 293-derived scFv demonstrating superior sensitivity. Computational chemistry analyses indicated that ASP-99 and ASN-102 residues in the scFv play key roles in antibody recognition, and the hydroxyl group near the benzene ring of the target molecule is critical for in antibody binding. Furthermore, we enhanced the scFv's biosensing sensitivity using an HCR-CRISPR/Cas12a amplification strategy in a streptavidin-based immunoassay. In the dual-step amplification process, detection limits for CAP in the HCR and HCR-CRISPR/Cas12a stages were significantly reduced to 55.23 pg/mL and 3.31 pg/mL, respectively. These findings introduce an effective method for developing CAP-specific scFv antibodies and also propose a multi-amplification strategy to increase immunoassay sensitivity. Additionally, theoretical studies also offer valuable guidance in CAP hapten design and genetic engineering for antibody modification.

Microfabrication of engineered Lactococcus lactis biocarriers with genetically programmed immunorecognition probes for sensitive lateral flow immunoassay of antibiotic in milk and lake water.

Micro/nanomaterials display considerable potential for increasing the sensitivity of lateral flow immunoassay (LFIA) by acting as 3D carriers for both antibodies and signals. The key to achieving high detection sensitivity depends on the probe's orientation on the material surface and its multivalent biomolecular interactions with targets. Here, we engineer Lactococcus lactis as the bacterial microcarrier (BMC) for a multivalent immunorecognition probe that was genetically programmed to display multifunctional components including a phage-screened single-chain variable fragment (scFv), an enhanced green fluorescent protein (eGFP), and a C-terminal peptidoglycan-binding domain (AcmA) anchored on BMC through the cell wall peptidoglycan. The innovative design of this biocarrier system, which incorporates a lab-on-a-chip microfluidic device, allows for the rapid and non-destructive self-assembly of the multivalent scFv-eGFP-AcmA@BMC probe, in which the 3D structure of BMC with a large peptidoglycan surface area facilitates the precisely orientated attachment and immobilization of scFv-eGFP-AcmA. This leads to a remarkable fluorescence aggregation amplification effect in LFIA, outperforming a monovalent 2D scFv-eGFP-AcmA probe for florfenicol detection. By designing a portable sensing device, we achieved an exceptionally low detection limit of 0.28 pg/mL and 0.21 pg/mL for florfenicol in lake water and milk sample, respectively. The successful microfabrication of this biocarrier holds potential to inspire innovative biohybrid designs for environment and food safety biosensing applications.

Synbiotic Combination between Lactobacillus paracasei VL8 and Mannan-Oligosaccharide Repairs the Intestinal Barrier in the Dextran Sulfate Sodium-Induced Colitis Model by Regulating the Intestinal Stem Cell Niche.

Previously, Lactobacillus paracasei VL8, a lactobacillus strain isolated from the traditional Finnish fermented dairy product Viili, demonstrated immunomodulatory and antibacterial effects. The prebiotic mannan-oligosaccharide (MOS) further promoted its antibacterial activity and growth performance, holding promise for maintaining intestinal health. However, this has not been verified in vivo. In this study, we elucidated the process by which L. paracasei VL8 and its synbiotc combination (SYN) with MOS repair the intestinal barrier function in dextran sodium sulfate (DSS)-induced colitis mice. SYN surpasses VL8 or MOS alone in restoring goblet cells and improving the tight junction structure. Omics analysis on gut microbiota reveals SYN's ability to restore Lactobacillus spp. abundance and promote tryptophan metabolism. SYN intervention also inhibits the DSS-induced hyperactivation of the Wnt/ß-catenin pathway. Tryptophan metabolites from Lactobacillus induce intestinal organoid differentiation. Co-housing experiments confirm microbiota transferability, replicating intestinal barrier repair. In conclusion, our study highlights the potential therapeutic efficacy of the synbiotic combination of Lactobacillus paracasei VL8 and MOS in restoring the damaged intestinal barrier and offers new insights into the complex crosstalk between the gut microbiota and intestinal stem cells.

Effect of different thermal processing methods on sensory, nutritional, physicochemical and structural properties of Penaeus vannamei.

The aim of this study was to investigate the effect of different thermal processing methods on the nutritional and physicochemical qualities of Penaeus vannamei. Three different thermal processing methods, namely, drying (DS, 120 °C/40 min), steaming (SS, 100 °C/2 min), and microwaving (MS, 600 W/2 min) were used to treat the shrimps. Low-field nuclear magnetic resonance data indicated that fixed water was the main component of Penaeus vannamei. The ratio of fatty acids in MS and DS samples was more in line with the FAO/WHO recommended health requirements; The myofibrillar protein carbonyl group increased, whereas sulfhydryl content decreased after thermal processing, indicating that the proteins were oxidized by thermal processing. The magnitude of oxidation is: MS > SS > DS. Different thermal processing methods can exert great influence on color texture and nutrition to Penaeus vannamei, which can provide a theoretical knowledge for consumers to choose the appropriate processing method.

Improvement in the gelling properties of myofibrillar protein from the razor clam (Sinonovacula constricta) through phosphorylation and structural characterization of the modified protein.

This study investigated the modification of myofibrillar protein (MP) from the razor clam through phosphorylation by using various phosphate salts, namely, sodium tripolyphosphate (STPP), sodium trimetaphosphate (STMP), sodium polyphosphate (STTP) and sodium pyrophosphate (TSPP), and their mechanisms of action for functional and gelling properties. Fourier transform infrared spectrometry (FTIR) showed that MP introduced phosphate groups during phosphorylation; these phosphates changed the secondary structure. Moreover, MP after phosphorylation led to an increase in solubility, which was more evident in the case of TSPP phosphorylation, leading to the improvement of gel properties. Therefore, TSPP was the phosphate with the best gel properties in the modification of MP, showing the highest phosphorus content, which resulted in better gelling properties owing to its relatively shorter chains. These results showed that phosphate was able to improve protein cross-linking through ion interactions and electrostatic interactions, which ultimately improved the gelling properties of the razor clam protein.

Prediction and validation of enzymatic degradation of aflatoxin M1: Genomics and proteomics analysis of Bacillus pumilus E-1-1-1 enzymes.

Aflatoxins are a class of highly toxic mycotoxins. Aflatoxin M1 (AFM1) is hydroxylated metabolite of aflatoxin B1, having comparable toxicity, which is more commonly found in milk. In this study, the whole genome sequencing of Bacillus pumilus E-1-1-1 isolated from feces of 38 kinds of animals, having aflatoxin M1 degradation ability was conducted. Bacterial genome sequencing indicated that a total of 3445 sequences were finally annotated on 23 different cluster of orthologous groups (COG) categories. Then, the potential AFM1 degradation proteins were verified by proteomics; the properties of these proteins were further explored, including protein molecular weight, hydrophobicity, secondary structure prediction, and three-dimensional structures. Bacterial genome sequencing combined with proteomics showed that eight genes were the most capable of degrading AFM1 including three catalases, one superoxide dismutase, and four peroxidases to clone. These eight genes with AFM1 degrading capacity were successfully expressed. These results indicated that AFM1 can be degraded by Bacillus pumilus E-1-1-1 protein and the most degrading proteins were oxidoreductases.

Marine Chitosan-Oligosaccharide Ameliorated Plasma Cholesterol in Hypercholesterolemic Hamsters by Modifying the Gut Microflora, Bile Acids, and Short-Chain Fatty Acids.

This study evaluated the cholesterol-alleviating effect and underlying mechanisms of chitosan-oligosaccharide (COS) in hypercholesterolemic hamsters. Male hamsters (n = 24) were divided into three groups in a random fashion, and each group was fed one particular diet, namely a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), and an HCD diet substituting 5% of the COS diet for six weeks. Subsequently, alterations in fecal bile acids (BAs), short-chain fatty acids (SCFAs), and gut microflora (GM) were investigated. COS intervention significantly reduced and increased the plasma total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels in hypercholesteremic hamsters. Furthermore, Non-HDL-C and total triacylglycerols (TG) levels were also reduced by COS supplementation. Additionally, COS could reduce and increase food intake and fecal SCFAs (acetate), respectively. Moreover, COS had beneficial effects on levels of BAs and GM related to cholesterol metabolism. This study provides novel evidence for the cholesterol-lowering activity of COS.

Monosaccharide-induced glycation enhances gelation and physicochemical properties of myofibrillar protein from oyster (Crassostrea gigas).

Glycation offers a promising potential to improve protein gelling properties in food industries. Therefore, the study was aimed to illustrate the effect of five monosaccharides (erythrose-aldotetrose, xylose-aldopentose, glucose-aldohexose, galactose-aldohexose, and fructose-ketohexose) with different carbon numbers and structure on the structure-gelling relationship of myofibrillar protein (MP) from oyster (Crassostrea gigas). Results showed that monosaccharides significantly increased the glycation degree of MP by increasing sulfhydryl content, forming stable tertiary conformation and decreasing surface hydrophobicity. Moreover, the gel properties of MP like gel strength, water holding capacity, water mobility were improved by alleviating aggregation including the increase of solubility and the decrease of particle sizes. Oyster MP glycated by glucose (aldohexose) possessed the optimal gel properties. Molecular docking simulation showed that hydrogen bonds and hydrocarbon bonds were the mainly non-covalent binding modes. The study will provide a theoretical basis for oyster protein glycation and expand its application on food gel.

Effects of ultrasound-assisted high temperature-pressure treatment on the structure and allergenicity of tropomyosin from clam (Mactra veneriformis).

Tropomyosin (TM) is the major allergen in clams. This study aimed to evaluate the effects of ultrasound-assisted high temperature-pressure treatment on the structure and allergenicity of TM from clams. The results showed that the combined treatment significantly affected the structure of TM-converting the α-helix to ß-sheet and random coil, and decreasing the sulfhydryl group content, surface hydrophobicity, and particle size. These structural changes caused the unfolding of the protein, disrupting and modifying the allergenic epitopes. The significant reduction in the allergenicity of TM was approximately 68.1% when treated with combined processing (P < 0.05). Notably, an increase in the content of the relevant amino acids and a smaller particle size accelerated the penetration of the enzyme into the protein matrix, resulting in strengthening the gastrointestinal digestibility of TM. These results prove that ultrasound-assisted high temperature-pressure treatment has great potential in reducing allergenicity, benefiting the development of hypoallergenic clam products.

Amino acid regulated citrus pectin-based emulsion stability mediated by pH.

BACKGROUND: Citrus residuals are rich in nutrients like pectin, essential oil, and amino acids, which are wasted in the food industry. Moreover, citrus components often coexist with amino acids during emulsion preparation and application. RESULTS: Adding glutamic or arginine after emulsification resulted in a stable emulsion compared with adding them before emulsification. Adding glycine before or after emulsification had no effect on the emulsion stability. Emulsion stability was improved by adding glutamic acid at pH 6. Ionic interaction and hydrogen bonding were the main forms of bonding. The rhamnogalacturonan II domain was the potential binding site for the amino acids. CONCLUSIONS: The emulsions prepared by adding acidic amino acids or basic amino acids after emulsification were stable relative to those in which the amino acids were added before emulsification. However, the order in which neutral amino acids were added did not affect the emulsion stability after storage for 7 days. With an increase in the pH level, droplet size increased and emulsion stability decreased. All the results could be attributed to changes in the structure and properties of citrus pectin, as well as the interaction between citrus pectin and amino acids. This study may expand the application of citrus-derived emulsions in the food industry. © 2023 Society of Chemical Industry.

Protocols for extraction and purification of rutin from leafy by-products of asparagus (Asparagus officinalis) and characterization of the purified product.

Valorization of asparagus leafy by-products as a potential source of rutin through selected extraction and purification protocols was investigated. Protocol resulting in the highest extraction yield was first selected. Crude extract was subject to purification via multiple liquid-liquid back extraction using ethanol, methanol or water as a solvent; selection of the most appropriate purification solvent was made based on rutin solubility. The proposed purification protocol yielded yellow-color crystals, which were characterized by fluorescence microscopy, Fourier-transform infrared spectroscopy and liquid chromatography-mass spectrometry to confirm them as rutin. Purity of rutin was confirmed by ultra-performance liquid chromatography at 97.6%; yield of the purified rutin was determined to be 78.2%. The remaining rutin (21.8%) was found in the liquids collected at various stages of purification; such liquids could be recycled using the same purification process. The proposed protocols are simple, yet effective for rutin extraction and purification from asparagus leafy by-products.