Jihong Hung,Xing Lyu,Aimei Lio,Yi Zho,Yinchen Hou,Wenjing Chen,Cnrui Yng

a College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

b Henan Cooperativity Medical Science and Technology Research Institute Co.,Ltd,Luoyang 471000,China

Keywords:

Fermentation

CWECM

Functional proteins

Proteomics

ABSTRACT

Compound probiotics were used to ferment compound wheat embryo Chinese medicine(CWECM)at 37°C for a given period,and the fermentation end point was determined based on pH value.The full spectrum of the proteins within the fermentation supernatant was determined by ion mass spectrometry,and then the fermentation was analyzed by proteomics.The difference of functional protein of CWECM was analyzed before and after fermentation.Additionally,the changes in flavor of peptides were examined.Our results indicated that when the fermentation was performed for 96 h,the pH value became stable over time,which it shifted from an initial 3.98±0.04 to a stable 2.78±0.04.Proteomicbased analysis of the fermentation supernatant indicated that the fermentation of the probiotics increased.The amount of functional proteome and protein within the supernatant increased from 60 proteomes and 136 proteins before fermentation to 70 proteomes and 149 proteins post fermentation.These proteins were primarily members of the Ubiquitin and Histone families.These families contain more specific peptides after fermentation,and the coverage was reduced.Analysis of the unique P0C512 protein after fermentation indicated that this protein is an unstable,3Deffect columnar protein.We also found that probiotic fermentation can reduce the proportion of bitter peptides within fermentation broth from 47.49%to 36.98%,and the proportion of sweet peptides increased after fermentation from 35.29%to 51.01%.Based on testing,we concluded that fermentation of CWECM by compound probiotics can reduce the pH of the CWECM solution,increase the number of functional proteomes and proteins,improve the taste of CWECM solutions,and improve the therapeutic efficacy of CWECM.

1.Introduction

Traditional Chinese medicine (TCM) is advantageous in that it is a multi-component medicine that can affect a number of therapeutic targets[1-4],and it possesses a long history of culture and clinical treatment uses.Bioinformatics analyses using proteomics-based technology are commonly employed to generate large data sets that can be used to reveal functional protein molecules and their physiological regulatory mechanisms,and these research methods are widely used for functional food examination,facilitation of health product development,and functional verification[5-7].Ji et al.[8]studied the immunomodulatory effects of WGG on cyclophosphamide-induced immunosuppressive mice,where they explored the immunological activity of WGG.Their results indicated that WGG could significantly reduce spleen and thymus immunosuppression(P ＜0.01)and could alleviate cyclophosphamide-induced damage to the spleen and thymus.Kuo et al.[9]found that CRM-CL/LIP inhibited the expression of phosphorylated p38(p-p38),phosphorylated c-Jun N-terminal kinase(p-JNK),and p-tau protein at serine 202 and prevented neurodegeneration of SK-N-MC cells.In addition,NGF-CL/LIP could enhance the quantities of p-neurotrophic tyrosine kinase receptor type 1 and p-extracellular signal-regulated kinase 5 for neuronal rescue.Zhang et al.[10]showed that the anticarcinogenic activities of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1(LFWGE).Ismail et al.[11]utilized chemical proteomics analysis technology to successfully identify 124 proteins that bind to artemisinin.Taken together,these findings indicate that proteomics-based technology is of great significance for the analysis and identification of proteins[12].

In the field of biomedicine,few studies have examined the combination of wheat embryo and traditional Chinese medicine(CWECM)in regard to improving the efficacy of traditional Chinese medicine and the utilization rate of wheat embryo.Here,CWECM was fermented using the preferred compound probiotic bacteria strain,and a Q-Exactive(QE)mass spectrometer was used for qualitative identification of protein using a proteomicsbased method.In regard to the fermentation of CWECM,changes in the composition of functional proteins were observed,and the taste of CWECM was improved by changes in the concentration of bitter peptides.

2.Materials and methods

2.1.Materials and reagents

To create compound probiotics,Lactobacillus plantarum,L.acidophilus,and Bifidobacterium(obtained from the laboratory of this research group)were mixed at specific proportions.CWECM was provided by Henan Qinglian(Da Health Industry Co.,Ltd.),and MRS medium was purchased from Zhengzhou Seamus Biotechnology Co.,Ltd.Deionized water was used for all tests,and it was purchased from Hongfa Water Marketing Department,Central Plains Region.

2.2.Fermentation of CWECM

CWECM powder(20 g)was added into a 250 mL Erlenmeyer flask at a 1:4 ratio with deionized water.This mixture was sterilized at 85 °C for 30 min,0.5%activated compound probiotic liquid was then added after cooling at the aseptic workstation,and the cooled mixture was then sealed with a sealing film and placed in a 37 °C constant temperature shaker for fermentation.The pH of the fermentation broth was measured at 1,6,12,24,48,96,and 120 h using a pH meter(Ray-Magnetic PHS-3E type,Shanghai INESA Scientific Instrument Co.,Ltd.).The fermentation was considered complete when the pH became stable.

2.3.Determination of protein spectrum of fermentation supernatant

The mass spectrometry process was slightly modified according to the previous method[13-15],and our mass spectroscopy process followed a number of steps.First,enzymatic hydrolysis was performed,where after reduction and alkylation,trypsin(W/W=1:50)was added,and the mixture was hydrolyzed at 37 °C for 20 h.Next,mass spectrometry analysis was performed.For this analysis,solution A consisted of a 0.1%formic acid aqueous solution,and B solution was a 0.1%formic acid solution(acetonitrile 84%).The chromatographic column used for this analysis was 95%A liquid at equilibrium.The sample was sampled from the automatic sampler to the Trap column under a 0.5 h gradient.Finally,mass spectrometry data detailing the fragments of peptides and whole peptides was collected.The ratio between mass and charge was determined from 20 fragment maps(Mass Spectrometry 2 scan)that were collected after each full scan.All the experiments were performed in three replicates.

2.4.Software and database

UniProt(https://www.uniprot.org/)was used as the protein analysis network database,and the secondary structure of proteins was analyzed using Predict Protein:(http://www.predictprotein.org/).The homologous modeling website SWISS-MODEL server(https://swissmodel.expasy.org/)was also used for data analysis.

2.5.Proteomic data analysis

Three parallel values were taken from each test site.Origin 8.5 software was used to process the drawing of the test data,and the Mascot 2.2 software was used to search the corresponding database for the raw file of the mass spectrometry test.Based on these analytic approaches,identified protein results were obtained.The search parameters are presented in Table 1.

3.Results and discussions

3.1.Change in pH value during fermentation

Compound probiotics contain a relatively high amount of lactic acid bacteria.Measuring the fermentation status of probiotics using pH can also indicate if the fermentation process is normal.pH values of the CWECM after fermentation by compound probiotics are provided inFig.1.After 96 h,the pH decreased slowly over time,and its value ultimately stabilized.This stabilization indicated that the fermentation process was complete at 96 h.The pH decreased from an initial value of 3.98 ±0.04 to a final value of 2.78 ±0.04.

Table 1 Database retrieval parameter table.

3.2.Determination of the protein spectrum of the fermentation supernatant

As indicated in Fig.2,the protein peaks of the supernatant before and after fermentation are primarily concentrated within the 8-20 min elution window,and the relative elution peaks and the abundance are both elevated,indicating that the peptides present in the samples are diverse and highly complex.Among them,the protein peaks of Fig.2A is higher than that of Fig.2B,which indicates that the content of polypeptide in the supernatant decreases after fermentation,which may be due to the decomposition of macromolecular proteins in CWECM into small molecular peptides.

3.3.Identification and analysis of proteome and peptides within fermentation liquor

We next identified and analyzed the proteomics,proteins,and peptide molecules using QE mass spectrometry and tandem mass spectrometry.The proteins obtained by reverse-transferring the peptides are arranged in order of reliability from low to high,and the proteins with higher conservation or homology are classified into the same proteome.The specific peptides determine the reliability of the analysis,and when the specific peptides ≥2,this confirms the analytical significance and allows for an accurate determination of the number of proteomes,proteins,and polypeptide molecules.As indicated in Table 2,the total number of proteins present in the supernatant before fermentation was 136,the total proteome was 60,and the fragment with specific peptide ≥2 accounted for 10.00%.The total number of proteins present in the supernatant after fermentation was 149,and the total proteome was 70.After fermentation,the total number of proteins increased by 13,the proteomes increased by 10,and the specific peptides ≥2 accounted for 12.86%.This was 2.86%higher than that measured before fermentation,and the specific peptide value was 4,indicating that the fermentation changes the macromolecular peptide into a small molecular peptide.After fermentation,the macromolecular proteins within the CWECM are altered into small molecule peptides,allowing for higher absorbance within the body.Additionally,the coverage of the peptide segment is correspondingly reduced.

Fig.1.Variation in pH value during fermentation.

Fig.2.Protein spectrum of the fermentation culture medium.

3.4.Analysis of functional proteins before and after fermentation

According to the comparison of protein content before and after fermentation and to the reference database,30 groups of proteomes that included 62 proteins existed before fermentation that were unique to the supernatant,and after fermentation,40 groups of proteomes that included 75 proteins existed that were unique to the supernatant.There were 30 groups of the same proteome in the centrifugation before and after fermentation,and these included 74 proteins.There were also 2 groups in the consensus protein group with more specific peptides(≥2),and these included 20 major proteins that are members of the ubiquitin(Table 3)and histone families(Table 4).Specific peptides ≥2 were used as analytical indicators,and there were 2 groups of unique proteomes within the centrifuge after fermentation that included 4 proteins.The main components are shown in Table 5.

Ubiquitin is a small protein found in all eukaryotes and in most eukaryotic cells.Its main function is to label proteins for degradation by the 26S proteasome.Ubiquitin can also label transmembrane proteins and can participate in the transport of proteins by membranes.A typical ubiquitin chains play an important role in cell signaling,endocytosis,DNA damage repair,and regulation of the NF-κB pathway.When polyubiquitin exists as free polyubiquitin,it can exert different effects such as activation of protein kinases and subsequent signaling pathways.Ubiquitin-like proteins are involved in the selective degradation of ATP-dependent cellular proteins,maintain chromatin structure,regulate gene expression,modulate the stress response,and influence ribosomal biosynthesis.

Table 2 Changes in the proteome and peptides before and after fermentation.

Table 3 Ubiquitin family of proteins before and after fermentation.

Table 4 Proteome before and after fermentation-histone family.

Histones are the core component of nucleosomes,and DNA accessibility is regulated by a series of complex post-translational modifications of histones(also known as histone coding and nuclear small-weight plastics).Nucleosomes encapsulate and compress DNA into chromatin,thus limiting the accessibility of DNA to other intracellular molecules that could use the DNA as a template.Therefore,histones play an important role in transcriptional regulation,DNA repair,DNA replication,and chromosomal stability.

RuBisCO catalyzes two reactions,including the carboxylation of Dribulose 1,5-bisphosphate,which is the primary event in carbon dioxide fixation,and the oxidative fragmentation of the pentose substrate required forthe photorespiration process.Both reactions occur simultaneously and compete for the same active site.This enzyme is the key enzyme in glycolysis that catalyzes the first step of the pathway by converting Dglyceraldehyde 3-phosphate(G3P)into 3-phospho-D-glyceroyl phosphate,and it is essential for the maintenance of cellular ATP levels and carbohydrate metabolism.

Table 5 Proteome with specific peptide ≥2 in centrifuge after fermentation.

3.5.Functional structure analysis of P0C512 protein

The primary sequence of P0C512 was retrieved from the UNIPROT database using P0C512 as the ID.According to the amino acid sequence of P0C512,the relative molecular weight is 52,881.14 Da and the isoelectric point is 6.22.The protein is comprised of 477 amino acids,and the highest glycine (Gly) content is 9.6%,and the alanine (Ala)content is 9.4%.This protein contains 60 negatively charged residues(Asp+ Glu),and it possesses 54 positively charges residues (Arg and Lys).The extinction coefficient of this protein when measured at 280 nm in water is 69,955,and it possesses a half-life of 30 h with a protein instability coefficient of 43.50.As an unstable parameter value below 40 is the standard for stabilizing proteins,it is likely that P0C512 is an unstable protein.The aliphatic index for this protein was 77.34,and the average hydrophilicity was 0.283.

The P0C512 sequence information was uploaded to the Predict Protein website for protein secondary structure prediction(Fig.3).The loop type accounted for 53.46%of protein structure,and the spiral type accounted for 33.75%.The folding type of secondary structure was present in the smallest amount at only 12.79%,indicating that these structures are unstable and generally correspond to protein areas where the function is not conserved.

The advanced structure of protein P0C512 was simulated using the online analysis tool SWISS-MODEL.We observed that the sequence discrimination rate using template protein 5iu0.1.A(X-ray,resolution 1.50 Å)was 0.71,the similarity of protein sequence was 0.6,and the coverage rate was 1.00.Therefore,this was selected as the template protein for P0C512(Fig.4).

The tertiary structure of entire P0C512 protein possesses a square structure,and the overall 3D effect is cylindrical.The structural analysis indicates that the three-level structural model is relatively stable.

3.6.Effect of fermentation on flavor peptide

Bitter peptides,sweet peptides,and sour peptides are collectively referred to as flavor peptides,and are mainly derived from fermentation,aging processes,or hydrolysis processing of foods[16,17].When food enters the oral cavity,oligopeptides(collectively referred to as flavor peptides)or amino acids possessing various flavors bind to different receptors on human taste bud cells,and different tastes can be perceived through neurotransmission[18].As illustrated in Table 6,during progression of the fermentation process,the total number of flavor peptides contained in the extract of the compound wheat germ increases from 179 to 192,and the bitter peptides are reduced to 71 from the original 85.The sweet peptides also increase after fermentation from 54 to 82,and the number of sour peptides did not change.Taken together,our findings indicate that fermentation not only reduced the bitterness of the traditional Chinese wheat germ,but also increased the sweetness and improved the taste and flavor of the edible medicine.

4.Conclusions

In this study,the functional proteins present in CWECM fermented by compound probiotics were analyzed by a proteome-based method.Our results demonstrated that after fermentation with compound probiotics,the macromolecular proteins in CWECM were transformed into small molecular peptides or small molecular substances,the quantity of functional proteins was increased,the molecular weight of large proteins was reduced,and the original bitter taste of traditional Chinese medicine was improved.Taken together,these changes will allow for easier consumption and absorbance of active ingredients within the human body.Our current study provides only a preliminary analysis of CWECM protein after fermentation.Future studies examining traditional processing technology differences,the underlying pharmacology of this medicine,and the biochemical mechanisms are still required.

Fig.3.Secondary structure of P0C512.

Fig.4.Structural simulation of P0C512.

Table 6 Contents and proportions of different flavor peptides before and after fermentation.

Conflicts of Interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgment

This work was supported by Centaline Thousand Talents Program Centaline Scholars Funding Program(No.192101510004),the Natural Science Foundation of Henan Province (No.182300410056),and Green Manufacturing 2025(Ministry of Industry and Information Technology[2018]No.272-3).