Germination Increases the Glucomoringin Content in Moringa Sprouts via Transforming Tyrosine.

Moringa seeds are an excellent dietary source of phytochemicals (i.e., glucosinolates, GSLs; isothiocyanates, ITCs) with health-beneficial effects. Although numerous studies have been conducted on moringa seeds, the effect of germination on the regulation of GSLs remains scarcely explored. The present study investigated the dynamic changes of GSLs in moringa seeds during germination (at 25, 30, and 35 °C for 6 days in the dark) through an untargeted metabolomics approach and compared the antioxidant capacity of ungerminated and germinated moringa seeds. Our results showed that germination significantly increased the total GSL content from 150 (day 0) to 323 µmol/g (35 °C, day 6) on a dry weight (DW) basis, especially glucomoringin (GMG), the unique glucosinolate in moringa seeds, which was significantly upregulated from 61 (day 0) to 149 µmol/g DW (35 °C, day 4). The upregulation of GMG corresponded to the metabolism of tyrosine, which might be the initial precursor for the formation of GMG. In addition, germination enhanced the total ITC content from 85 (day 0) to 239 µmol SE/g DW (35 °C, day 6), indicating that germination may have also increased the activity of myrosinase. Furthermore, germination remarkably increased the total phenolic content (109-507 mg GAE/100 g DW) and antioxidant capacity of moringa seeds. Our findings suggest that moringa sprouts could be promoted as a novel food and/or ingredient rich in GMG.

3D Porous Edible Scaffolds from Rye Secalin for Cell-Based Pork Fat Tissue Culturing.

Cellular agriculture holds hope for a sustainable alternative to conventional meat, yet multiple technical challenges remain. These include the large-scale production of edible scaffolds and culturing methods for fat tissues, which are key to meat texture, flavor, and nutritional values. Herein. we disclose our method in the facile fabrication of sponge-like plant protein scaffolds by applying commercial sugar cubes as highly permeable templates. The prepared secalin scaffolds feature a high porosity of 85-90%, fully interconnected pores, and high water stability. The mechanical properties of scaffolds could be tuned by varying sugar-to-protein weight ratio and post-water annealing treatment. Moreover, murine preadipocytes (3T3-L1) and porcine adipose-derived stem cells (ADSCs) readily infiltrate, adhere, proliferate, and differentiate on the secalin scaffolds to develop a fat tissue morphology. A cultured fat tissue was produced by culturing porcine ADSCs for 12 days, which remarkably resembles conventional porcine subcutaneous adipose tissue in appearance, texture, flavor, and fatty acid profiles. The research demonstrates the great potential of sponge-like secalin scaffolds for cultured fat tissue production.

The thermal degradation of glucomoringin and changes of phenolic compounds in moringa seed kernels during different degrees of roasting.

The effect of heat treatment on the abundant bioactive compounds in moringa seed kernels (MSKs) during different degrees of roasting remains sparingly explored despite the flour of roasted MSKs has been incorporated into the human diet (e.g., cakes, cookies, and burgers) as a substitute to enrich the nutritional content. Therefore, we investigated the impacts of different roasting conditions (e.g., temperature and duration) on bioactive compounds (e.g., glucosinolates (GSLs), phenolic acids and alkaloids) and antioxidant capacity of MSKs. Our results showed that light and medium roasting increased the glucomoringin (GMG, the main GSL in MSKs) content from 43.7 (unroasted MSKs) to 69.7-127.3 µmol/g MSKs (dry weight), while excessive/dark roasting caused thermally-induced degradation of GMG (trace/undetectable level) in MSKs, resulting in the formation of various breakdown products (e.g., thiourea, nitrile, and amide). In addition, although roasting caused a significant reduction of some phenolic compounds (e.g., gallic, chlorogenic, p-coumaric acids, and trigonelline), other phenolic acids (e.g., caffeic and ferulic acids) and alkaloids (e.g., caffeine, theobromine, and theophylline) remarkably increased after roasting, which may contribute to the enhanced total phenolic content (up to 2.9-fold) and antioxidant capacity (up to 5.8-fold) of the roasted MSKs.

Reclaiming Agriceuticals from Sweetpotato (Ipomoea batatas [L.] Lam.) By-Products.

Sweetpotato (SP, Ipomoea batatas [L.] Lam.) is a globally significant food crop known for its high nutritional and functional values. Although the contents and compositions of bioactive constituents vary among SP varieties, sweetpotato by-products (SPBs), including aerial parts, storage root peels, and wastes generated from starch processing, are considered as excellent sources of polyphenols (e.g., chlorogenic acid, caffeoylquinic acid, and dicaffeoylquinic acid), lutein, functional carbohydrates (e.g., pectin, polysaccharides, and resin glycosides) or proteins (e.g., polyphenol oxidase, ß-amylase, and sporamins). This review summarises the health benefits of these ingredients specifically derived from SPBs in vitro and/or in vivo, such as anti-obesity, anti-cancer, antioxidant, cardioprotective, and anti-diabetic, evidencing their potential to regenerate value-added bio-products in the fields of food and nutraceutical. Accordingly, conventional and novel technologies have been developed and sometimes combined for the pretreatment and extraction processes aimed at optimising the recovery efficiency of bioactive ingredients from SPBs while ensuring sustainability. However, so far, advanced extraction technologies have not been extensively applied for recovering bioactive compounds from SPBs except for SP leaves. Furthermore, the incorporation of reclaimed bioactive ingredients from SPBs into foods or other healthcare products remains limited. This review also briefly discusses current challenges faced by the SPB recycling industry while suggesting that more efforts should be made to facilitate the transition from scientific advances to commercialisation for reutilising and valorising SPBs.

Authentication of ten distinctive triterpenoids in Antrodia cinnamomea serves as a crucial aspect for ensuring the quality control of associated nutraceutical products.

Edible mushroom Antrodia cinnamomea is distinctive for its use in many health supplement products in relieving of diverse health-related conditions. A. cinnamomea is known for its rich array of bioactive secondary metabolites, predominantly terpenoids, that possess anti-inflammatory properties. Despite the abundance of these compounds, only some compounds have demonstrated notable anti-inflammatory activity. Moreover, there is a lack of established quality control methods specifically tailored to the active constituents of these products. Consequently, there is a great need for the development of precise and effective quality control methods for A. cinnamomea-based products, targeting their active components to ensure the consistency and reliability of these products in harnessing their anti-inflammatory potential. Herein we report a quantitative HPLC method for better evaluating the quality of A. cinnamomea based dietary supplements. Based on their bioactivities, we selected ten benchmark compounds, i. e. antcin K, (25S)-antcin H, (25R)-antcin H, (25R)-antcin C, (25S)-antcin C, (25R)-antcin A, 15α-acetyl-dehydrosulphurenic acid, versisponic acid D, dehydroeburicoic acid, and eburicoic acid and developed and validated a HPLC-UV method for quantification of these compounds simultaneously with high sensitivity, linearity and range, precision, and accuracy. Furthermore, we applied our method to quantify the commercially available A. cinnamomea containing supplements and found that the quality of these supplements varies greatly with only one product containing good amount of the active compounds. Our method provides a needed solution to quality control problem of the highly priced A. cinnamomea food and nutraceutical products that show great variety and inconsistency.

Protective Effect of Selenium-enriched Peptide from Cardamine violifolia on Ethanol-induced L-02 Hepatocyte Injury.

In this study, we investigated the protective effect of selenium (Se)-enriched peptide isolated from Cardamine violifolia (SPE) against ethanol-induced liver injury. Cell proliferation assays show that different concentrations of SPE protect human embryonic liver L-02 cells against ethanol-induced injury in a dose-dependent manner. Treatment with 12 µmol/L Se increases the cell survival rate (82.44%) and reduces the release of alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, and apoptosis rate. SPE treatment with 12 µmol/L Se effectively reduces the concentration of intracellular reactive oxygen species and increases the contents of intracellular superoxide dismutase (51.64 U/mg), catalase (4.41 U/mg), glutathione peroxidase (1205.28 nmol/g), and glutathione (66.67 µmol/g), thereby inhibiting the effect of ethanol-induced oxidative damage. The results of the transcriptomic analysis show that the glutathione metabolism and apoptotic pathway play significant roles in the protection of L-02 hepatocytes by SPE. Real-time qPCR analysis shows that SPE increases the mRNA expression of GPX1 and NGFR. The results of this study highlight the protective effects of SPE against ethanol-induced liver injury.

A newly synthesized flavone avoids COMT-catalyzed methylation and mitigates myocardial ischemia/reperfusion injury in H9C2 cells via JNK and P38 pathways.

Objectives: Luteolin is a flavone that provides defense against myocardial ischemia/reperfusion (I/R) injury. However, this compound is subjected to methylation mediated by catechol-O-methyltransferase (COMT), thus influencing its pharmacological effect. To synthesize a new flavone from luteolin that avoids COMT-catalyzed methylation and find out the protective mechanism of LUA in myocardial I/R injury. Materials and Methods: Luteolin and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were used to synthesize the new flavone known as LUAAPH-1 (LUA). Then, the myocardial ischemia/reperfusion injury cell model was established using H9c2 cells to detect the effect in myocardial ischemia/reperfusion regulation and to identify the underlying mechanism. Results: Pretreatment with LUA (20 µmol/l) substantially increased cell viability while reducing cell apoptosis rate and caspase-3 expression induced by I/R, and the protective effect of LUA on cell viability was stronger than diosmetin, which is the major methylated metabolite of luteolin. In addition, intracellular reactive oxygen species (ROS) production and calcium accumulation were both inhibited by LUA. Furthermore, we identified that LUA markedly relieved the promotive effects of I/R stimulation upon JNK and p38 phosphorylation. Conclusion: LUT pretreatment conveys significant cardioprotective effects after myocardial I/R injury, and JNK and p38 MAPK signaling pathway may be involved.

Investigation of selenium and selenium species in Cardamine violifolia using in vitro digestion coupled with a Caco-2 cell monolayer model.

Inadequate Se intake can enhance vulnerability to certain health risks, with supplementation lessening these risks. This study investigated the bioavailability of Se and Se species in five Se compounds and in Se-rich Cardamine violifolia using in vitro digestion coupled with a Caco-2 cell monolayer model, which enabled the study of Se transport and uptake. Translocation results showed that SeCys2 and MeSeCys had high translocation rates in C. violifolia leaves (CVLs). The uptake rate of organic Se increased with time, and MeSeCys exhibited a higher uptake rate than that for SeCys2 and SeMet. The translocation mechanisms of SeMet, Se(IV), and Se(VI) were passive transport, whereas those of SeCys2 and MeSeCys were active transport. The bioavailability of organic Se was higher than that of inorganic Se, with a total Se bioavailability in CVLs of 49.11 %. This study would provide a theoretical basis for the application of C. violifolia in the functional food.

Bioproduction of Rare d-Allulose from d-Glucose via Borate-Assisted Isomerization.

d-Allulose is a low-calorie functional rare sugar with excellent processing suitability and unique physiological efficacy. d-Allulose is primarily produced from d-fructose through enzymatic epimerization, facing the constraints of a low conversion yield and high production cost. In this study, a double-enzyme cascade system with tetraborate-assisted isomerization was constructed for the efficient production of d-allulose from inexpensive d-glucose. With the introduction of sodium tetraborate (STB), capable of forming complexes with diol-bearing sugars, the conversion yield of d-allulose from d-glucose substantially escalated from the initial 17.37% to 44.97%. Furthermore, d-allulose was found to exhibit the most pronounced binding affinity for STB with an association constant of 1980.51 M-1, notably surpassing that of d-fructose (183.31 M-1) and d-glucose (35.37 M-1). Additionally, the structural analysis of the sugar-STB complexes demonstrated that d-allulose reacted with STB via the cis 2,3-hydroxyl groups in the α-furanose form. Finally, the mechanism underlying STB-assisted isomerization was proposed, emphasizing the preferential formation of an allulose-STB complex that effectively shifts the isomerization equilibrium to the allulose side, thereby resulting in high yield of d-allulose. Such an STB-facilitated isomerization system would also provide a guidance for the cost-effective synthesis of other rare sugars.

The Synergistic Effect of Compound Sugar with Different Glycemic Indices Combined with Creatine on Exercise-Related Fatigue in Mice.

In this study, a compound sugar (CS) with different glycemic index sugars was formulated via hydrolysis characteristics and postprandial glycemic response, and the impact of CS and creatine emulsion on exercise-related fatigue in mice was investigated. Thirty-five C57BL/6 mice were randomly divided into five groups to supply different emulsions for 4 weeks: initial emulsion (Con), glucose emulsion (62 mg/10 g MW glucose; Glu), CS emulsion (62 mg/10 g MW compound sugar; CS), creatine emulsion (6 mg/10 g MW creatine; Cr), and CS and creatine emulsion (62 mg/10 g MW compound sugar, 6 mg/10 g MW creatine, CS-Cr). Then, the exhaustion time of weight-bearing swimming and forelimb grip strength were measured to evaluate the exercise capacity of mice, and some fatigue-related biochemical indexes of blood were determined. The results demonstrated that the ingestion of CS significantly reduced the peak of postprandial blood glucose levels and prolonged the energy supply of mice compared to ingesting an equal amount of glucose. Mouse exhaustion time was 1.22-fold longer in the CS group than in the glucose group. Additionally, the supplementation of CS increased the liver glycogen content and total antioxidant capacity of mice. Moreover, the combined supplementation of CS and creatine increased relative forelimb grip strength and decreased blood creatine kinase activity. The findings suggested that the intake of CS could enhance exercise capacity, and the combined supplementation of CS and creatine has a synergistic effect in improving performance.

Chemical Composition of Thyme (Thymus vulgaris) Extracts, Potential Inhibition of SARS-CoV-2 Spike Protein-ACE2 Binding and ACE2 Activity, and Radical Scavenging Capacity.

Water and ethanol extracts of dried thyme (Thymus vulgaris) were analyzed for chemical composition, inhibition of the SARS-CoV-2 spike protein-ACE2 interaction, inhibition of ACE2 activity, and free radical scavenging capacity. Thirty-two compounds were identified in water extract (WE) and 27 were identified in ethanol extract (EE) of thyme through HPLC-MS. The WE (33.3 mg/mL) and EE (3.3 mg/mL) of thyme inhibited the spike protein-ACE2 interaction by 82.6 and 86.4%, respectively. The thyme WE at 5 mg/mL inhibited ACE2 activity by 99%, and the EE at 5 mg/mL inhibited ACE2 by 65.8%. Total phenolics were determined to be 38.9 and 8.8 mg of GAE/g in WE and EE, respectively. The HO• scavenging capacities were 1121.1 and 284.4 µmol of TE/g in WE and EE, respectively. The relative DPPH• scavenging capacities were 126.3 µmol TE/g in WE and 28.2 µmol TE/g in EE. The ABTS•+ scavenging capacities were 267.1 µmol TE/g in WE and 96.7 µmol TE/g in EE. The results suggested that the thyme extract could be potentially used to prevent SARS-CoV-2 infection and mitigate the complications from the infection.

Non-extractable polyphenols from blue honeysuckle fruit pomace with strong antioxidant capacity: Extraction, characterization, and their antioxidant capacity.

The aim of this study was to investigate a more practical method for obtaining non-extractable polyphenols (NEPPs) from blue honeysuckle fruit pomace. Three methods, namely acid, alkaline, and enzymatic hydrolysis, were utilized to extract NEPPs. The findings indicated that alkaline hydrolysis was the most effective method for releasing NEPPs, which demonstrated higher levels of total flavonoid content (TFC) and total phenolic content (TPC) from blue honeysuckle fruit pomace. Additionally, higher TPC and TFC levels were related to a stronger antioxidant capacity. Qualitative and quantitative analysis using HPLC-HR-TOF-MS/MS revealed that acid hydrolysis resulted in a greater concentration of certain phenolic acids, while alkaline hydrolysis yielded a higher concentration of flavonoids, and enzymatic hydrolysis produced a wider range of phenolic compositions. Despite the fact that enzymatic hydrolysis is considered a gentler method, the researchers concluded that alkaline hydrolysis was the most appropriate method for obtaining NEPPs from blue honeysuckle fruit pomace.

Trypsin-treated chickpea protein hydrolysate enhances the cytoaffinity of microbeads for cultured meat application.

Cultured meat is believed to be a promising alternative to conventional meat production that can reduce environmental impacts, animal suffering, and food safety risks. However, one of the major challenges in producing cultured meat is to provide suitable microcarriers that can support cell attachment, proliferation, and differentiation. In this study, we developed novel microcarriers based on chickpea protein hydrolysates functionalized with trypsin. These microcarriers exhibited superior cytoaffinity and proliferation for various types of cultured cells, including C2C12, porcine myoblasts, chicken satellite cells, and 3T3-L1. Moreover, these microcarriers enabled cell differentiation into muscle or fat cells under appropriate conditions. We propose that trypsin treatment enhances the cytoaffinity of chickpea protein hydrolysates by exposing lysine and arginine residues that can interact with cell surface receptors. Our results suggest that chickpea protein hydrolysate functionalized microcarrier is a promising substrate for cultured meat production with cost-effectiveness and scalability.

Comprehensive structural analysis of polyphenols and their enzymatic inhibition activities and antioxidant capacity of black mulberry (Morus nigra L.).

In this paper, the structures of polyphenols and their bioactivity of black mulberry (Morus nigra L.) cv. 'Heisang No. 1' were comprehensively analyzed. The 11 anthocyanins and 20 non-anthocyanin phenolic compounds were identified and quantified by liquid chromatography high-resolution time-of-flight mass spectrometry (LC-HR-TOF/MS2). The cyanidin-3-glucoside and cyanidin-3-rutinoside were the major anthocyanins in the black mulberry. In addition, the black mulberry showed potent antioxidant capacity as assessed by DPPH, ABTS, and FRAP assays. Black mulberry anthocyanins exhibited stronger inhibition activities against α-amylase, α-glucosidase, and lipase compared to non-anthocyanin polyphenols, with IC50 values of 1.10, 4.36, and 9.18 mg/mL, respectively. The total anthocyanin content of black mulberry crude extracts and anthocyanins was 570.10 ± 77.09 and 1278.23 ± 117.60 mg C3GE/100 g DW, respectively. Black mulberry may be a rich source of polyphenols, natural antioxidants, and effective antidiabetic substances with great potential in the food industry.

Machine learning and 3D bioprinting.

48With the growing number of biomaterials and printing technologies, bioprinting has brought about tremendous potential to fabricate biomimetic architectures or living tissue constructs. To make bioprinting and bioprinted constructs more powerful, machine learning (ML) is introduced to optimize the relevant processes, applied materials, and mechanical/biological performances. The objectives of this work were to collate, analyze, categorize, and summarize published articles and papers pertaining to ML applications in bioprinting and their impact on bioprinted constructs, as well as the directions of potential development. From the available references, both traditional ML and deep learning (DL) have been applied to optimize the printing process, structural parameters, material properties, and biological/mechanical performance of bioprinted constructs. The former uses features extracted from image or numerical data as inputs in prediction model building, and the latter uses the image directly for segmentation or classification model building. All of these studies present advanced bioprinting with a stable and reliable printing process, desirable fiber/droplet diameter, and precise layer stacking, and also enhance the bioprinted constructs with better design and cell performance. The current challenges and outlooks in developing process-material-performance models are highlighted, which may pave the way for revolutionizing bioprinting technologies and bioprinted construct design.

Antioxidant Activities of Dihydromyricetin Derivatives with Different Acyl Donor Chain Lengths Synthetized by Lipozyme TL IM.

Dihydromyricetin (DHM) is a phytochemical with multiple bioactivities. However, its poor liposolubility limits its application in the field. In this study, DHM was acylated with different fatty acid vinyl esters to improve its lipophilicity, and five DHM acylated derivatives with different carbon chain lengths (C2-DHM, C4-DHM, C6-DHM, C8-DHM, and C12-DHM) and different lipophilicity were synthesized. The relationship between the lipophilicity and antioxidant activities of DHM and its derivatives was evaluated with oil and emulsion models using chemical and cellular antioxidant activity (CAA) tests. The capacity of DHM derivatives to scavenge 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS+•) was similar to that of DHM, except for C12-DHM. The antioxidant activity of DHM derivatives was lower than that of DHM in sunflower oil, while C4-DHM exhibited better antioxidant capacity in oil-in-water emulsion. In CAA tests, C8-DHM (median effective dose (EC50) 35.14 µmol/L) exhibited better antioxidant activity than that of DHM (EC50: 226.26 µmol/L). The results showed that in different antioxidant models, DHM derivatives with different lipophilicity had various antioxidant activities, which has guiding significance for the use of DHM and its derivatives.

Study on the mechanism of various exogenous proteins with different inhibitions on wheat starch digestion: From the distribution behaviors of protein in the starch matrix.

This study aimed to compare the effect of various exogenous proteins on wheat starch (WS) digestion and assess the relevant mechanisms based on the distribution behaviors of exogenous proteins in the starch matrix. Rice protein (RP), soy protein isolate (SPI), and whey protein isolate (WPI) all effectively suppressed the rapid digestion of WS but with different modes. RP increased the slowly digestible starch content, while SPI and WPI increased the resistant starch content. Fluorescence images showed that RP aggregated and competed for effective space with starch granules, while SPI and WPI formed continuous network structures among the starch matrix. These distribution behaviors endowed different reductions in starch digestion by influencing the gelatinization and ordered structure of starch. Pasting and water mobility results suggested all exogenous proteins inhibited the water migration and swelling of starch. Simultaneously, X-ray diffraction and Fourier transform infrared spectroscopy analysis showed that exogenous proteins improved the ordered structures of starch. RP had a more significant effect on the long-term ordered structure, while SPI and WPI had a more effective effect on the short-term ordered structure. These findings will enrich the theory of exogenous protein inhibiting starch digestion and inspire the applications in low-glycemic index food.

Targeting the Na+/K+ ATPase DR-region with DR-Ab improves doxorubicin-induced cardiotoxicity.

Doxorubicin (DOX) is a potent chemotherapeutic drug for various cancers. Yet, the cardiotoxic side effects limit its application in clinical uses, in which ferroptosis serves as a crucial pathological mechanism in DOX-induced cardiotoxicity (DIC). A reduction of Na+/K + ATPase (NKA) activity is closely associated with DIC progression. However, whether abnormal NKA function was involved in DOX-induced cardiotoxicity and ferroptosis remains unknown. Here, we aim to decipher the cellular and molecular mechanisms of dysfunctional NKA in DOX-induced ferroptosis and investigate NKA as a potential therapeutic target for DIC. A decrease activity of NKA further aggravated DOX-triggered cardiac dysfunction and ferroptosis in NKAα1 haploinsufficiency mice. In contrast, antibodies against the DR-region of NKAα-subunit (DR-Ab) attenuated the cardiac dysfunction and ferroptosis induced by DOX. Mechanistically, NKAα1 interacted with SLC7A11 to form a novel protein complex, which was directly implicated in the disease progression of DIC. Furthermore, the therapeutic effect of DR-Ab on DIC was mediated by reducing ferroptosis by promoting the association of NKAα1/SLC7A11 complex and maintaining the stability of SLC7A11 on the cell surface. These results indicate that antibodies targeting the DR-region of NKA may serve as a novel therapeutic strategy to alleviate DOX-induced cardiotoxicity.

Polyphenols in twenty cultivars of blue honeysuckle (Lonicera caerulea L.): Profiling, antioxidant capacity, and α-amylase inhibitory activity.

The polyphenols extracted from 20 blue honeysuckle cultivars were comprehensively characterized and quantified by HPLC-DAD and HPLC-ESI-QTOF-MS2 analyses and evaluated for antioxidant capacity (ABTS, DPPH, FRAP) and α-amylase inhibitory activity. The 17 anthocyanins and 59 non-anthocyanin phenolics were characterized. Among them, cyanidin-3-glucoside, quercetin-3-galactoside, myricetin-3-galactoside, and 3-caffeoylquinic acid were the major polyphenols. These polyphenols not only contributed to the antioxidant capacity, but were also good α-amylase inhibitors. 'Lanjingling' showed the strongest antioxidant capacity evaluated by FRAP, while 'CBS-2' and '14-13-1' showed the strongest antioxidant capacity evaluated by ABTS and DPPH. All the twenty cultivars showed α-amylase inhibitory activity, and the IC50 values ranged from 0.12 ± 0.01 to 0.69 ± 0.02 mg/mL. 'Lanjingling' showed the most potent α-amylase inhibitory activity. Additionally, principal component analysis indicated that Lonicera. caerulea subsp. emkuyedao bred in Japan differed markedly in phenolics and bioactivity compared to the other four subspecies bred in China and Russia.