Structural and functional properties of whey protein isolate-inulin conjugates prepared with ultrasound or wet heating method.

BACKGROUND: Whey protein isolate (WPI) generally represents poor functional properties such as thermal stability, emulsifying activity and antioxidant activity near its isoelectric point or high temperatures, which limit its application in the food industry. The preparation of WPI-polysaccharide covalent conjugates based on Maillard reaction is a promising method to improve the physical and chemical stability and functional properties of WPI. In this research, WPI-inulin conjugates were prepared through wet heating method and ultrasound method and their structural and functional properties were examined. RESULTS: In conjugates, the free amino acid content was reduced, the high molecular bands were emerged at sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), new C-N bonds were formed in Fourier-transform infrared (FTIR) spectroscopy, and fluorescence intensity was reduced compared with WPI. Furthermore, the result of circular dichroism (CD) spectroscopy also showed that the secondary structure of conjugates was changed. Conjugates with ultrasound treatment had better structural properties compared with those prepared by wet heating treatment. The functional properties such as thermal stability, emulsifying activity index (EAI), emulsion stability (ES) and antioxidant activity of conjugates with wet heating treatment were significantly improved compared with WPI. The EAI and ES of conjugates with ultrasound treatment were the highest, but the thermal stability and antioxidant activity were only close to that of the conjugates with wet heating treatment for 2 h. CONCLUSION: This study revealed that WPI-inulin conjugates prepared with ultrasound or wet heating method not only changed the structural characteristics of WPI but also could promote its functional properties including thermal stability, EAI, ES and antioxidant activity. © 2024 Society of Chemical Industry.

Isolation, purification, and mass spectrometry identification of the enzyme involved in citrus flavor (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolytica.

BACKGROUND: (+)-Nootkatone is a highly valuable sesquiterpene compound that can be used as an aromatic in the food industry because of its grapefruit flavor and low sensory threshold. The unconventional yeast Yarrowia lipolytica has many unique physical and chemical properties, metabolic characteristics, and genetic structure, which has aroused the interest of researchers. Previous research showed that Y. lipolytica possesses the ability to transform the sesquiterpene (+)-valencene to (+)-nootkatone. The aim of this study was to isolate, purify, and identify the enzyme involved in the (+)-valencene bioconversion to (+)-nootkatone by Y. lipolytica. RESULTS: In this study, ultrasonic-assisted extraction, ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography were used to separate and purify the enzyme involved in the (+)-valencene bioconversion by Y. lipolytica. The protein was identified as aldehyde dehydrogenase (ALDH) (gene0658) using sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. The ALDH had the highest activity when the pH value was 6.0 and the temperature was 30 °C. The activity of ALDH was significantly stimulated by ferrous ions and inhibited by barium, calcium, and magnesium ions. CONCLUSION: This is the first time that ALDH was found to participate in (+)-valencene biotransformation by Y. lipolytica. It may be involved in regulating the microbial transformation of (+)-valencene to (+)-nootkatone through redox characteristics. This study provides a theoretical basis and reference for the biological synthesis of citrus flavor (+)-nootkatone. © 2023 Society of Chemical Industry.

Effect of different ways of ingesting orange essential oil on blood immune index and intestinal microflora in mice.

BACKGROUND: Studies have found that the addition of plant essential oils to feed had a positive effect on intestinal microflora and immunity in mice. However, the effect of different ways of ingestion of orange essential oil on mice has seldom been reported. In the present study, we investigated the effects of ingestion of orange essential oil by gavage, sniffing and feeding on intestinal microflora and immunity in mice. RESULTS: The results obtained showed that a low concentration of essential oil feeding significantly increased the spleen index of mice (P < 0.05). The effect of different ways of ingestion on the thymus index, immunoglobulin G and immunoglobulin M of mice was not significant (P > 0.05). High and medium concentrations of essential oil feeding increased the level of interleukin-2 in mice (P < 0.05). H+ K+ -ATPase activity was significantly increased in mice fed with gavage and different concentrations of essential oil feed compared to the control group (P < 0.05). The analysis of the results of the microflora in the cecum and colon of mice indicated that the medium concentration of essential oil feeding group and the sniffing group significantly changed the structure of the flora and increased the diversity of the intestinal microflora. All three essential oil ingestion methods increased the abundance of Bacteroidetes and Lactobacillus in the intestine of mice. CONCLUSION: Compared with gavage and feeding, sniffing had a significant effect on immunoglobulins in mice. All the three ingestion methods could affect the intestinal microflora of mice and increase the abundance of Lactobacillus. © 2022 Society of Chemical Industry.

Identification of functional genes associated with the biotransformation of limonene to trans-dihydrocarvone in Klebsiella sp. O852.

BACKGROUND: Natural dihydrocarvone has been widely used in the food, cosmetics, agrochemicals and pharmaceuticals industries because of its sensory properties and physiological effects. In our previous study, Klebsiella sp. O852 was shown to be capable of converting limonene to trans-dihydrocarvone with high catalytic efficiency. Thus, it was essential to identify and characterize the functional genes involved in limonene biotransformation using genome sequencing and heterologous expression. RESULTS: The 5.49-Mb draft genome sequence of Klebsiella sp. O852 contained 5218 protein-encoding genes. Seven candidate genes participating in the biotransformation of limonene to trans-dihydrocarvone were identified by genome analysis. Heterologous expression of these genes in Escherichia coli BL21(DE3) indicated that 0852_GM005124 and 0852_GM003417 could hydroxylate limonene in the six position to yield carveol, carvone and trans-dihydrocarvone. 0852_GM002332 and 0852_GM001602 could catalyze the oxidation of carveol to carvone and trans-dihydrocarvone. 0852_GM000709, 0852_GM001600 and 0852_GM000954 had high carvone reductase activity toward the hydrogenation of carvone to trans-dihydrocarvone. CONCLUSION: The results obtained in the present study suggest that the seven genes described above were responsible for converting limonene to trans-dihydrocarvone. The present study contributes to providing a foundation for the industrial production of trans-dihydrocarvone in microbial chassis cells using synthetic biology strategies. © 2021 Society of Chemical Industry.

Separation and purification of nootkatone from fermentation broth of Yarrowia lipolytica with high-speed counter-current chromatography.

Nootkatone is an important functional sesquiterpene, which can be obtained by the biotransformation of valencene. It is increasingly important because of its pleasant citrus aroma and physiological effects. Yarrowia lipolytica is beneficial for biotechnology applications and has ability to transform valencene to nootkatone. High-speed counter-current chromatography (HSCCC) was used to isolate and purify the product of nootkatone in this study. The suitable two-phase solvent system was selected and the optimum separation conditions were determined. The partition coefficients of nootkatone and the separation factor between nootkatone and valencene were considered as the indexes. The results showed that there were numerous products during the transformation of valencene by Yarrowia lipolytica, and the content of nootkatone was 13.75%. The obtained nootkatone was separated by HSCCC with a solvent system n-hexane/methanol/water (5/4/1, v/v). The final purity of nootkatone was 91.61 ± 0.20% and the elution time was 290-310 min. The structure of nootkatone was identified by gas chromatography-mass spectrometry (GC-MS), infrared spectrum and nuclear magnetic resonance hydrogen spectroscopy (1H NMR). This was the first report on the separation of nootkatone from the fermentation broth by HSCCC. This study proved that HSCCC could be used as an effective method to separate and purify the nootkatone from valencene transformed by Yarrowia lipolytica with n-hexane/methanol/water (5/4/1, v/v).

Advances on (+)-nootkatone microbial biosynthesis and its related enzymes.

(+)-Nootkatone is an important functional sesquiterpene and is comprehensively used in pharmaceutical, cosmetic, agricultural and food flavor industries. However, (+)-nootkatone is accumulated trace amounts in plants, and the demand for industry is mainly met by chemical methods which is harmful to the environment. The oxygen-containing sesquiterpenes prepared using microbial methods can be considered as "natural." Microbial transformation has the advantages of mild reaction conditions, high efficiency, environmental protection, and strong stereoselectivity, and has become an important method for the production of natural spices. The microbial biosynthesis of (+)-nootkatone from the main precursor (+)-valencene is summarized in this paper. Whole-cell systems of fungi, bacteria, microalgae, and plant cells have been employed. It was described that the enzymes involved in the microbial biosynthesis of (+)-nootkatone, including cytochrome p450 enzymes, laccase, lipoxygenase, and so on. More recently, the related enzymes were expressed in microbial hosts to heterologous produce (+)-nootkatone, such as Escherichia coli, Pichia pastoris, Yarrowia lipolytica, and Saccharomyces cerevisiae. Finally, the development direction of research for realizing industrialization of microbial transformation was summarized and it provided many options for future improved bioprocesses.

Screening a Strain of Klebsiella sp. O852 and the Optimization of Fermentation Conditions for Trans-Dihydrocarvone Production.

Flavors and fragrances have high commercial value in the food, cosmetic, chemical and pharmaceutical industries. It is interesting to investigate the isolation and characterization of new microorganisms with the ability to produce flavor compounds. In this study, a new strain of Klebsiella sp. O852 (accession number CCTCC M2020509) was isolated from decayed navel orange (Citrus sinensis (L.) Osbeck), which was proved to be capable of converting limonene to trans-dihydrocarvone. Besides, the optimization of various reaction parameters to enhance the trans-dihydrocarvone production in shake flask was performed for Klebsiella sp. O852. The results showed that the yield of trans-dihydrocarvone reached up to 1 058 mg/L when Klebsiella sp. O852 was incubated using LB-M medium for 4 h at 36 °C and 150 rpm, and the biotransformation process was monitored for 36 h after adding 1680 mg/L limonene/ethanol (final ethanol concentration of 0.8% (v/v)). The content of trans-dihydrocarvone increased 16 times after optimization. This study provided a basis and reference for producing trans-dihydrocarvone by biotransformation.

Effect and mechanism of high-fat diet on the preference for sweeteners on mice.

BACKGROUND: Male Kunming mice were divided into a normal diet group (control group) and a high-fat diet group (HF group) (185 g·kg-1 protein, 600 g·kg-1 fat and 205 g·kg-1 carbohydrate). After 8 weeks' feeding, behavioral indicators and biochemical indicators in serum were determined. The double-bottle preference experiment was used to study the preferences of mice for five sweeteners. The monoamine neurotransmitter content, gene expression related to dopamine (DA), and opioid receptors were also determined. RESULTS: The body weight of the mice in the HF group increased significantly (P < 0.05) after 36 days compared with the control group. The feed intake of the HF group increased sharply in the first 12 days, and then it became basically unchanged. The preference of the HF group for all of the five sweeteners was highly significantly lower (P < 0.01) than that of the control group. Depression-related behavior was observed in the HF group mice. The triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDLC) content in the HF group were very much higher (P < 0.01) than those of the control group. The gene expression related to DA and opioid receptor in the HF group was significantly lower than that of the control group, except for preproenkephalin (PENK). CONCLUSIONS: In summary, this study suggested that a long-term high-fat diet could result in a decrease in the preference for sweeteners and could result in a state of reward hypofunction in mice. © 2020 Society of Chemical Industry.

Effect of short-term intake of four sweeteners on feed intake, solution consumption and neurotransmitters release on mice.

This study focused on the effect of short-term intake of sweeteners on feed intake, solution consumption and neurotransmitters release on mice. The results showed that the free drinking of 10 mM sucralose solution, 100 mM maltose solution, 3 mM saccharin solution and 3 g/L stevioside solution for 32 days will not affect the normal development of the body weight and feed intake of the mice. The consumption of maltose solution was significantly higher than that of the other sweeteners. The leptin and insulin levels increased significantly after the short-term intake of these four sweeteners. The dopamine (DA) content in the whole brain of the mice increased significantly only in the maltose group. These results indicate that the short-term intake of the preferred concentrations of maltose, stevioside, sucralose and saccharin will not affect the body weight and feed intake of the mice. Mice prefer maltose solution to other sweeteners solutions. The 100 mM maltose solution and 3 mM saccharin solution could result in the oxidative stress on mice after 32 days' short-term intake. Compared with other sweeteners, only sugars that could be broken down into small molecules of glucose might have a positive effect on dopamine levels.

Effects of different sweeteners on behavior and neurotransmitters release in mice.

Four natural sweeteners (sucrose, stevioside, maltose and xylitol) and six artificial sweeteners (acesulfame, sucralose, aspartame, cyclamate, saccharin and neotame) were used to study the effects of different sweeteners on the behavior and neurotransmitter release of mice with two-bottle preference experiments. The results showed that very significant preference behavior for 8% sucrose solution, 0.3% stevioside solution, 10 mM acesulfame, 10 mM sucralose and 10 mM aspartame solutions (p < 0.01) was observed on mice. Long-term exposure of sucrose solution and acesulfame solution can affect the behavioral indicators such as solution consumption, feed intake, body weight and the release of neurotransmitters in mice. The solution consumption and the release of neurotransmitters were significantly greater (p < 0.05) than that of the control group (water group), but there was no significant difference in feed intake. The acesulfame-A and acesulfame-B groups had no significant difference on the consumption of solution and feed intake, but there was significant difference in the release of neurotransmitters. The result also showed that different sweetener solutions with similar sweetness had the same effect on the neurotransmitters release, and it can be inferred that mice have an addictive behavioral characteristic to sucrose.

Effects of orange essential oil on intestinal microflora in mice.

BACKGROUND: The intestinal microbiota has a wide variety of functions in the host. A positive effect of plant extract on intestinal microbiota in animals has been reported. However, the effect of orange essential oil and its components limonene, linalool and citral on intestinal microflora in mice has seldom been reported. The effects of intragastric administration of orange essential oil and limonene, linalool and citral on intestinal microflora and biochemical indexes in mice were studied. RESULTS: The effect of essential oil, linalool and citral on immune organ index (spleen and thymus index), IgM and IL-2 was not significant (P > 0.05). A significant increase (P < 0.05) of H+ K+ -ATPase activity, IgA, IgG, and IL-2 in the limonene group was observed when compared with the control group. Orange essential oil, limonene, linalool and citral could significantly reduce the content of short-chain fatty acids in the cecum and colon of mice. Principal coordinates analysis showed that intestinal bacterial structure of limonene group cecum and colon was apparently separated from other groups. The relative abundance of Lactobacillus in cecum and colon in essential oil, limonene, linalool and citral groups was higher than that in the control group. CONCLUSIONS: Orange essential oil and limonene, linalool and citral could affect the intestinal microflora of mice, and enhance the relative abundance of Lactobacillus. The intestinal bacterial structure of cecum and colon in the limonene group was quite different from other groups. This indicated a more obvious effect of limonene on intestinal bacteria, also resulting in significant changes in blood immune index and short-chain fatty acids in mice. © 2019 Society of Chemical Industry.

Changes of aroma compounds and qualities of freshly-squeezed orange juice during storage.

This study focused on the changes of physicochemical and microbiological properties and aroma compounds of freshly-squeezed orange juice during storage at different temperatures. Aroma compounds were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). The results showed that the total aerobic plate counts of orange juice stored at room temperature and 37 °C was far more than 4 °C. Totally 33 aroma compounds were determined in these orange juices. Significant differences on the aroma compounds in orange juices stored at different temperatures were observed in the present study. Most of the terpenes decreased at 4 °C after 15 days' storage, while 10 and 8 terpenes increased during storage at room temperature and 37 °C. α-Terpineol and p-vinylguaiacol were the only off-flavor compounds found in juice stored at 4 °C and room temperature at late storage respectively. While terpinen-4-ol, 4-ethylguaiacol and p-vinylguaiacol were found in juice stored at 37 °C at late storage. α-Terpineol was the only off-flavor compound found in orange juice stored at 4 °C.

Effect of olive oil on the preparation of nanoemulsions and its effect on aroma release.

The present study focused on the effect of olive oil on Ostwald ripening of flavor nanoemulsions. The release of the aroma compounds from the nanoemulsion system was also investigated. The results showed that the droplets size of the nanoemulsions decreased sharply first and then kept stable with the increase of Tween 80. The optimum surfactant/cosurfactant (Km) ratio was determined at 7:1. The average particle size of nanoemulsion was 39.22 nm. The polydispersity index (PDI) was 0.242 nm, and the particle size distribution was in the range of 20-150 nm at the optimum Km. The stability of the nanoemulsions was improved after the addition of olive oil, and it increased noticeably with the increase of olive oil. The addition of olive oil could help to stabilize the emulsions and hamper Ostwald ripening. All the 11 aroma compounds in the nanoemulsions were detected after 24-h storage. While only 5 aroma compounds were found after 48-h storage, and α-pinene and ß-myrcene were the only two aroma compounds detected after 72-h storage with low contents of 1.41 and 0.5 mg/L. The addition of olive oil inhibited the release of the aroma compounds from the nanoemulsion system. The released ethyl acetate was reduced by 48% after the addition of 10% olive oil. Significant decrease on the release of α-pinene and nonanal was observed after the addition of 3% olive oil. And the decrease was also observed on the release of ß-myrcene, D-limonene, α-terpineol, decanal and eugenol when the olive oil content was ≥ 5%. However, benzyl alcohol, ß-ionone and 1-octanol showed no significant changes with the increase of olive oil. This indicated that the addition of olive oil could provide greater retention of the aroma compounds in the nanoemulsions.

Proteins differentially expressed during limonene biotransformation by Penicillium digitatum DSM 62840 were examined using iTRAQ labeling coupled with 2D-LC-MS/MS.

This study focused on the differences in protein expression at various periods during limonene biotransformation by Penicillium digitatum DSM 62840. A total of 3644 protein-species were quantified by iTRAQ during limonene biotransformation (0 and 12 h). A total of 643 proteins were differentially expressed, 316 proteins were significantly up-regulated and 327 proteins were markedly down-regulated. GO, COG, and pathway enrichment analysis showed that the differentially expressed proteins possessed catalytic and binding functions and were involved in a variety of cellular and metabolic process. Furthermore, the enzymes involved in limonene transformation might be related to cytochrome P-450. This study provided a powerful platform for further exploration of biotransformation, and the identified proteins provided insight into the mechanism of limonene transformation.

Effect of Food Emulsifiers on Aroma Release.

This study aimed to determine the influence of different emulsifiers or xanthan-emulsifier systems on the release of aroma compounds. Solid-phase microextraction (SPME) and GC-MS were used to study the effects of varying concentrations of xanthan gum, sucrose fatty acid ester, Tween 80 and soybean lecithin on the release of seven aroma compounds. The effects of the emulsifier systems supplemented with xanthan gum on aroma release were also studied in the same way. The results showed varying degrees of influence of sucrose fatty acid ester, soybean lecithin, Tween 80 and xanthan gum on the release of aroma compounds. Compared with other aroma compounds, ethyl acetate was more likely to be conserved in the solution system, while the amount of limonene released was the highest among these seven aroma compounds. In conclusion, different emulsifiers and complexes showed different surface properties that tend to interact with different aroma molecules. The present studies showed that the composition and structure of emulsifiers and specific interactions between emulsifiers and aroma molecules have significant effects on aroma release.

Optimisation of α-terpineol production by limonene biotransformation using Penicillium digitatum DSM 62840.

BACKGROUND: In this study, (R)-(+)-limonene biotransformation using three fungal strains was compared. Penicillium digitatum DSM 62840 was distinguished for its capacity to transform limonene into α-terpineol with high regioselectivity. Growth kinetics in submerged liquid culture and the effects of growth phase and contact time on biotransformation were studied using this strain. Substrate concentration, co-solvent selection, and cultivation conditions were subsequently optimised. RESULTS: The maximum concentration of α-terpineol (833.93 mg L(-1)) was obtained when the pre-culture medium was in medium log-phase by adding 840 mg L(-1) substrate dissolved in ethanol and cultivation was performed at 24 °C, 150 rpm, and pH 6.0 for 12 h. Addition of small amounts of (R)-(+)-limonene (84 mg L(-1)) at the start of fungal log-phase growth yielded a 1.5-fold yield of α-terpineol, indicating that the enzyme was inducible. CONCLUSION: Among these three strains tested, P. digitatum DSM 62840 was proved to be an efficient biocatalyst to transform (R)-(+)-limonene to α-terpineol. Further studies revealed that the optimal growth phase for biotransformation was in the medium log phase of this strain. The biotransformation represented a wide tolerance of temperature; α-terpineol concentration underwent no significant change at 8-32 °C. The biotransformation could also be performed using resting cells.

Characteristics of ß-glucosidase from oranges during maturation and its relationship with changes in bound volatile compounds.

BACKGROUND: The hydrolysis of glycosidically bound volatile compounds can release potential aromas in oranges during maturation. ß-Glucosidase is the key enzyme that influences the hydrolysis of bound volatiles. In this study the changes in ß-glucosidase and bound volatile compounds in Jincheng oranges during maturation were investigated. The relationship between ß-glucosidase activity and bound volatiles was analyzed. RESULTS: The optimal temperature and pH of ß-glucosidase from Jincheng oranges were 40 °C and 5-6 respectively. Its Km and Vmax values were 0.61 mmol L(-1) and 0.009 U mg(-1) respectively. The activity of ß-glucosidase was strongly inhibited by Zn(2+), Fe(2+), Cu(2+), Ag(+), Hg(2+) and Fe(3+). ß-Glucosidase activity in pulp increased gradually during maturation, while that in peel first increased and then decreased in November. In total, 12 and 14 bound volatiles were found in pulp and peel respectively of this orange during maturation. CONCLUSION: The concentration of bound volatiles in pulp and peel decreased with the rise in ß-glucosidase activity in pulp and peel during maturation. This indicated that bound volatiles in Jincheng oranges were released during maturation owing to the increase in ß-glucosidase.

Inulin: properties and health benefits.

Inulin, a soluble dietary fiber, is widely found in more than 36 000 plant species as a reserve polysaccharide. The primary sources of inulin, include Jerusalem artichoke, chicory, onion, garlic, barley, and dahlia, among which Jerusalem artichoke tubers and chicory roots are often used as raw materials for inulin production in the food industry. It is universally acknowledged that inulin as a prebiotic has an outstanding effect on the regulation of intestinal microbiota via stimulating the growth of beneficial bacteria. In addition, inulin also exhibits excellent health benefits in regulating lipid metabolism, weight loss, lowering blood sugar, inhibiting the expression of inflammatory factors, reducing the risk of colon cancer, enhancing mineral absorption, improving constipation, and relieving depression. In this review paper, we attempt to present an exhaustive overview of the function and health benefits of inulin.

Genomic and transcriptomic analysis screening key genes for (+)-valencene biotransformation to (+)-nootkatone in Yarrowia lipolytica.

Yarrowia lipolytica is a kind of unconventional yeast, which is widely used in food industry because of its safety. (+)-Nootkatone, the ketone derivatives of (+)-valencene, possesses typical grapefruit aroma and is used as aromatics and medicines. It was found that Yarrowia lipolytica was an efficient biocatalyst for the transformation of (+)-valencene to (+)-nootkatone. Thus, it was meaningful to explore the genome features and the gene expression differences of strain Yarrowia lipolytica during (+)-valencene biotransformation, and to study the detailed bioconversion pathways. The results showed that the Yarrowia lipolytica genome was about 20.49 Mb, which encoded 6 137 protein coding genes. There were 1 167 differentially expressed genes (DEGs) in Y_V_36h ((+)-valencene-treated condition) compared to Y_36h ((+)-valencene-untreated blank). During biotransformation, the expression of genes related to the biosynthesis of secondary metabolites and most of ATP-binding cassette (ABC) transporters were significantly up-regulated. In addition, the expression of genes involved in energy metabolism decreased. Moreover, the enzymes participated in (+)-valencene biotransformation were inducible and they were inhibited by cytochrome P450 inhibitors. Several differentially expressed genes related to cytochrome P450 and dehydrogenase (gene2800, gene2911 and gene3152) were significantly up-regulated and might be responsible for converting (+)-valencene to (+)-nootkatone. The RT-qPCR experiment of ten DEGs were further verified and confirmed the reliability of transcriptome results. This study provided a basis for exploring the related genes and molecular regulatory mechanism of (+)-nootkatone biosynthesis from (+)-valencene by Yarrowia lipolytica.

Differential proteomic analysis of citrus flavor (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolytica.

Natural products (+)-nootkatone is an important sesquiterpene compound and is widely used in pharmaceutical, cosmetic, agricultural and food industries. The aim of this study was to analyze the differentially expressed proteins (DEPs) during citrus aroma compound (+)-valencene biotransformation to (+)-nootkatone by Yarrowia lipolyticaby with high-throughput LC-MS/MS. A total of 778 proteins were differentially expressed, 385 DEPs were significantly up-regulated and 393 DEPs were markedly down-regulated. It was found that the enzymes transformed (+)-valencene to (+)-nootkatone were mainly existed in yeast intracellular and precipitated under the condition of 30-40 % ammonium sulfate. Most DEPs involved in amino acid and fatty acid metabolism were down-regulated during (+)-valencene biotransformation. The DEPs related to the carbohydrate metabolism, energy metabolism and most of transporter proteins were significantly up-regulated. Furthermore, the key enzymes involved in (+)-valencene transformation might be related to cytochrome P450s (gene2215 and gene2911) and dehydrogenases (gene6493). This is the first time that proteomics was used to investigate the metabolism mechanism of Yarrowia lipolytica during (+)-valencene biotransformation. The proteomic analysis of Yarrowia lipolytica provided a foundation for the molecular regulatory mechanism in the biotransformation to (+)-nootkatone from (+)-valencene.