Exploring the modulatory role of bovine lactoferrin on the microbiome and the immune response in healthy and Shiga toxin‑producing E.coli challenged weaned piglets

Background Post-weaned piglets suffer from F18+Escherichia coli(E.coli)infections resulting in post-weaning diar-rhoea or oedema disease.Frequently used management strategies,including colistin and zinc oxide,have contrib-uted to the emergence and spread of antimicrobial resistance.Novel antimicrobials capable of directly interacting with pathogens and modulating the host immune responses are being investigated.Lactoferrin has shown promising results against porcine enterotoxigenic E.coli strains,both in vitro and in vivo.Results We investigated the influence of bovine lactoferrin(bLF)on the microbiome of healthy and infected weaned piglets.Additionally,we assessed whether bLF influenced the immune responses upon Shiga toxin-producing E.coli(STEC)infection.Therefore,2 in vivo trials were conducted:a microbiome trial and a challenge infection trial,using an F18+STEC strain.BLF did not affect theα-andβ-diversity.However,bLF groups showed a higher relative abundance(RA)for the Actinobacteria phylum and the Bifidobacterium genus in the ileal mucosa.When analysing the immune response upon infection,the STEC group exhibited a significant increase in F18-specific IgG serum levels,whereas this response was absent in the bLF group.Conclusion Taken together,the oral administration of bLF did not have a notable impact on theα-andβ-diversity of the gut microbiome in weaned piglets.Nevertheless,it did increase the RA of the Actinobacteria phylum and Bifi-dobacterium genus,which have previously been shown to play an important role in maintaining gut homeostasis.Furthermore,bLF administration during STEC infection resulted in the absence of F18-specific serum IgG responses.

Impact of an oligosaccharide-based polymer on the metabolic profiles and microbial ecology of weanling pigs experimentally infected with a pathogenic E.coli

Background Our previous study has reported that supplementation of oligosaccharide-based polymer enhances gut health and disease resistance of pigs infected with enterotoxigenic E.coli(ETEC)F18 in a manner similar to carbadox.The objective of this study was to investigate the impacts of oligosaccharide-based polymer or antibiotic on the host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18.Results Multivariate analysis highlighted the differences in the metabolic profiles of serum and colon digesta which were predominantly found between pigs supplemented with oligosaccharide-based polymer and antibiotic.The relative abundance of metabolic markers of immune responses and nutrient metabolisms,such as amino acids and carbohydrates,were significantly differentiated between the oligosaccharide-based polymer and antibiotic groups(q<0.2 and fold change>2.0).In addition,pigs in antibiotic had a reduced(P<0.05)relative abundance of Lachnospiraceae and Lactobacillaceae,whereas had greater(P<0.05)Clostridiaceae and Streptococcaceae in the colon digesta on d 11 post-inoculation(PI)compared with d 5 PI.Conclusions The impact of oligosaccharide-based polymer on the metabolic and microbial profiles of pigs is not fully understood,and further exploration is needed.However,current research suggest that various mechanisms are involved in the enhanced disease resistance and performance in ETEC-challenged pigs by supplementing this polymer.

Evaluating the Potential of Nitrofurantoin and Fosfomycin for E.coli UTIs: A Susceptibility Study

This study was designed to find the susceptibility of Nitrofurantoin and Fosfomycin among urinary isolates of Escherichia.coli.Four hundred(400)urine samples were collected for susceptibility of nitrofurantoin and fosfomycin among urinary isolates of E.coli.All indoor and outdoor patients'urinary samples yielded growth of E.coli.Mid-stream urine specimens were inoculated on blood agar and CLED agar and incubated at 35±2°C.Growth was observed,and Escherichia coli was identified by Gram staining,Catalase,Motility test and API 20E(Bio murex)as per standard procedure.Antimicrobial susceptibility testing of isolates for nitrofurantoin and fosfomycin was carried out by the modified Kirby-Bauer disc diffusion method according to CLSI guidelines ATCC 25922.E.coli was used as a quality control strain.A total of 400 samples were tested susceptibility of nitrofurantoin and fosfomycin among urinary isolates of E.coli during this period.A total of 400 samples yielded the growth of E.coli,out of which 178(44.5%)were male and 222(55.5%)were female samples.Among males,18(10%)were tolerant to nitrofurantoin,and 2(1.1%)were tolerant to fosfomycin.Among females,9(4.09%)were susceptible to nitrofurantoin while 6(2.72%)were susceptible to fosfomycin.Among age groups below 45 years old,6(4.76%)were tolerant to nitrofurantoin,and 2(1.58%)were sensitive to fosfomycin.Between 46-66 years old,4(2.81%)were sensitive to nitrofurantoin,and 3(2.11%)were sensitive to fosfomycin.Between 67-90 years old,17(12.87%)were sensitive to nitrofurantoin,and 4(3.03%)were tolerant to fosfomycin.Fosfomycin and nitrofurantoin showed good susceptibility in urinary isolates of E.coli and can be used empirically in our setup.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli

AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.

High-level expression of human calmodulin in E.coli and its effects on cell proliferation

Calmodulin (CaM), widely distributed in almost all eukaryotic cells, is a major intracellular calcium receptor responsible for mediating the Ca2 + signal to a multitude of different enzyme systems and is thought to play a vital role in the regulation of cell proliferative cycle[1，2]. Recently, many studies showed that CaM is also present in extracellular fluid such as cell culture media and normal body fluid and has been reported to stimulate proliferation in a range of normal and neoplastic cells, apparently acting as an autocrine growth factor[3-11]. In 1988, Crocker et al reported for the first time that addition of extracellular pure pig brain CaM could promote DNA synthesis and cell [7]proliferation in K562 human leukaemic lymphocytes[7].After that, more and more research was done on extracellular CaM and evidences demonstrated that extracellular CaM could also stimulate cell proliferation in normal human umbilical vein endothelial cells[5], keratinocytes[4], suspension-cultured cells of Angelica Dahurica, etc[6]. CaM is a monomeric protein of 148 amino acids that contains four homologous Ca2 + -binding domains. CaM has been highly conserved throughout the evolution. Only 1 out of 148 amino acids of human CaM is different from that of fish CaM. Complementary DNAs encoding rat, eel, chicken, human, and trypanosome CaM have been cloned.

An Improved Strategy for Efficient Expression and Purification of Soluble HIV-1 Tat Protein in E.coli

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Inducible Expression and Splicing of Candida Group I Ribozyme in E.coli

The Ca. LSU intron flanking a 129 bp cxon upstream and a fOO bp exondownstream was inserted into the lacZ gene on pRS426 to transform E. coli. Northern blot analysisand RT-PCR showed that splicing of Ca. LSU in E. coli is efficient upon inducible expression of theprecursor RNA, In contrast, co-lranscriptional self-splicing of the intron in vitro is much lessactive. Therefore, this E. coli splicing system can be used as a better model to investigate theeffect of the ribozyme inhibitors on Ca. LSUsplicing in living cell. We examined (he effects ofneomycin sulfatc and pentamidine on Ca. LSU splicing in E. coli, and found that these drugsdoes-dependently inhibit the intron splicing. However, heomycin is more potent than pentamidine inthis action.

Cloning and high-level expression of human interferon alpha-8 in E.coli

Human interferon alpha-8(IFN-α8) is an important cytokine with multiple biological functions.A genetically engineered strain, E. coli XL1-Blue/pBm, was constructed by DNA recombination technology and characterized by restriction analysis, DNA sequencing.

Expression of the human era cDNA in E.coli

Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.

Differential expression of bcl-2 and bax genes during the E.coli-induced apoptosis of human U937 cells

To explore the role of bcl-2 and bax genes in the apoptosis of human U937 cells induced by E.coli, flow cytometry assay with annexinⅤ-FITC/PI double staining was used to determine the condition of apoptosis, and the expressions of mRNA of bcl-2 and bax genes were assayed with RT-PCR. It was found that the apoptosis of human U937 cells could be induced by E.coli at various concentration ratios between cells and bacteria for 30 min in a dose-dependent manner. The apoptotic rates at cell/bacteria ratios of 0, 1∶5, 1∶10, 1∶20, 1∶50 and 1∶100 were 3.16%±0.90%, 9.46%±0.84%, 17.90%±1.41%, 35.59%±3.76%, 38.35%±7.12% and 55.07%±5.82% respectively. Also, there was a tendency of alterations in the expression levels of bcl-2 and bax genes with an increased expression level of bax gene and a reduced expression level of bcl-2 gene. It is concluded that E.coli can induce apoptosis in human U937 cells with a down-regulated expression of Bcl-2 and an up-regulated expression of Bax, and this might be related to the induction of apoptosis of the infected cell.

2种氟喹诺酮类抗生素与群体感应抑制剂对E.coli的联合毒性效应

抗生素滥用带来严重的细菌耐药性,威胁生态环境和人体健康。群体感应抑制剂(quorum sensing inhibitors,QSIs)作为一种理论上难以引发细菌耐药性的新型潜在抗生素替代品,被建议单独使用或与传统抗生素联合使用。因此,考察抗生素与QSIs联合作用效应及其作用机理对其在环境中可能产生的联合暴露风险评估具有重要的参考意义。以应用较广泛的2种氟喹诺酮类药物氧氟沙星(ofloxacin,OFL)、左氧氟沙星(levofloxacin,LEV)和1种新型抗菌剂群体感应抑制剂4-羟基-2,5-二甲基-3(2H)呋喃酮(4-hydroxy-2,5-dimethyl-3(2H)-furanone,HDMF)为研究对象,运用直接均分法和均匀设计射线法分别设计3个二元和1个三元混合物体系,每个体系包含5条具有不同组分浓度比的射线。应用时间毒性微板分析法测定3种药物及其混合物体系对大肠杆菌(Escherichia coli,E.coli)的毒性,应用拟合归零法分析混合物的毒性相互作用及相互作用强度,采用分子间对接技术来探讨可能存在的作用机理。结果表明,HDMF、OFL、LEV对E.coli均具有浓度、时间依赖毒性,以半数效应浓度负对数为毒性指标,3种药物在同一暴露时间毒性顺序:LEV>OFL>HDMF。3种药物的二元混合物体系相互作用类型有拮抗/协同作用,而三元混合物体系的作用类型为协同作用,且作用类型和强度受混合物组分、暴露时间和浓度影响。氟喹诺酮类药物混合物体系中,因药物竞争结合蛋白点位而呈现出拮抗作用。在氟喹诺酮类药物和QSIs混合体系中,QSIs会破坏细菌的生物膜,使氟喹诺酮类药物更容易接触细菌造成损伤,从而呈现协同作用。但随着暴露时间的延长,氟喹诺酮类药物会对DNA造成损伤进而减少QSIs作用蛋白的产生,呈现出拮抗作用。

大肠杆菌多酚氧化酶的分子克隆及异源高效表达

菜籽粕、棉籽粕等非粮饲料资源存在的酚类化合物芥子碱、棉酚等抗营养因子,严重影响了其饲喂价值。本课题组筛选出的一株大肠杆菌属芥子碱降解菌SDB2已被证实能通过分泌多酚氧化酶来降解芥子碱和棉酚,本试验旨在运用基因工程技术克隆SDB2多酚氧化酶基因,实现其高效表达,为SDB2多酚氧化酶在饲料工业上的规模化应用奠定基础。从大肠杆菌SDB2中克隆出多酚氧化酶基因,将其与质粒pET28a连接后转化至大肠杆菌BL21感受态细胞中培养,通过PCR鉴定及质粒酶切验证方法构建重组菌株,同时对重组克隆基因进行生物信息学分析。采用“2×2”交叉试验设计,以温度和诱导剂IPTG浓度两个影响因素诱导SDB2多酚氧化酶基因在重组菌株中高效表达,经SDS-PAGE和WB双重验证后,确立最佳诱导条件,最终纯化出目标酶蛋白。结果表明:(1)成功克隆SDB2多酚氧化酶基因,构建出异源表达重组菌株。(2)克隆出的SDB2重组多酚氧化酶基因含目标核苷酸741个,总编码氨基酸263个(含标签氨基酸20个),氨基酸组成的蛋白相对分子质量为28499.0,理论等电点为6.94,据此预测出SDB2重组多酚氧化酶三维结构模型。(3)确立SDB2多酚氧化酶异源高效表达的最佳诱导条件为温度37℃,IPTG浓度1 mmol,该条件下表达出大量可溶性酶蛋白,有利于工业化生产。本试验条件下,大肠杆菌SDB2多酚氧化酶成功实现了分子克隆及异源高效表达,为我国非粮饲料资源实现高效利用提供了技术参考。

电子商务快递末端配送模式优化研究

针对电子商务末端快递配送过程中车辆配送路径复杂和车辆利用率低等问题,设计了综合考虑车辆行驶距离和车辆装载率的多目标车辆路径优化模型,利用改进的非最优动态粒子群算法进行求解,并结合K-Means聚类来降低求解维度。通过改进学习因子和最优解更新策略,增强了粒子群算法的全局寻优能力,加快了收敛速度。最后,利用Solomon数据集中的R101样例验证了算法的有效性。结果表明:企业对用户进行合理归类后再规划车辆配送路径,可有效减少车辆行驶距离,提升车辆装载率,进而提升企业的配送效率,减少企业的运营成本,该研究可为电子商务环境下“最后一公里”配送模式的创新提供理论依据和决策支持。

溶血素 E基因原核表达载体的构建与蛋白诱导表达

目的构建大肠杆菌(Escherichia Coli,E.coli)溶血素E(Hemolysin E)基因的重组质粒pCZN1-hlyE,诱导表达目的蛋白。方法通过NCBI数据库检索获取E.coli Hemolysin E基因序列(NC_000913.3),采用基于PAS(PCR-based Accurate Synthesis)的全基因合成法,设计全长拼接引物,合成目的基因,并成功构建重组质粒pCZN1-hlyE。将构建好的pCZN1-hlyE重组质粒转入大肠杆菌TOP10菌株中,并用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Hemolysin E基因的表达,生成Hemolysin E毒力蛋白。结果经SDS-PAGE凝胶电泳,Western blot分析鉴定目的蛋白Hemolysin E分子量为28.72 kDa,与Hemolysin E基因核酸序列经DNAMAN软件分析所得蛋白分子量一致。结论被诱导表达的Hemolysin E蛋白存在于菌体裂解液上清中,并纯化制备重组蛋白。

禽致病性大肠杆菌HlyE蛋白的免疫原性研究

为评估禽致病性大肠杆菌(APEC)溶血素HlyE蛋白的免疫原性,本研究对APEC溶血素HlyE蛋白进行原核表达和纯化,并对表达的重组蛋白进行SDS-PAGE和western blot分析,将纯化蛋白利用透析袋在4℃透析后,利用血琼脂平板对其溶血活性进行检测。SDS-PAGE和western blot结果显示,表达并纯化到分子量约为36 ku的重组HlyE蛋白(rHlyE),经ND-2000超微量核酸蛋白测定仪测定纯化后蛋白浓度为0.65 mg/mL;溶血活性检测结果显示,rHlyE具有溶血活性。以透析后的rHlyE作为抗原,按照50μg/只的剂量免疫小鼠,对照组于相同时间点注射等量PBS,共免疫3次间隔14 d,并于首免后不同时间采血,采用间接ELISA方法检测两组小鼠血清特异性抗体水平,并于三免后18 d以2 LD_(50)的APEC菌液攻毒小鼠,观察7 d内小鼠的死亡情况;于首免后28 d剖杀各组小鼠取其脾脏制备脾淋巴细胞,采用流式细胞术分别检测CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞亚型比率,对rHlyE的免疫原性进行评估。间接ELISA检测结果显示,该蛋白能够诱导机体产生体液免疫应答,分泌高表达量的IgG抗体,抗体水平于三免后15 d达到最高水平。rHlyE免疫攻毒保护试验结果显示,免疫组小鼠基本无明显临床症状,7 d内存活率达80%;而对照组小鼠表现明显临床症状,于攻毒后3 d内全部死亡。流式细胞术结果显示,与对照组相比,免疫组小鼠的CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞亚型比率均升高。上述结果表明,rHlyE在大肠杆菌BL21中为部分可溶性表达,将其免疫小鼠后可诱导小鼠产生较高水平的体液免疫应答,并且可对小鼠产生较好的免疫保护效果。本研究明确了APEC HlyE蛋白的免疫原性,为APEC免疫保护蛋白的筛选以及疫苗研发提供了借鉴与参考。

重组E.coli工程菌高密度培养生产人源型胶原蛋白

Several technical parameters were studied during the fermentation of recombinant E.coli for the production of collagen-like biopolymer.The effects of dissolved oxygen as well as glucose concentration on fermentation were observed.The OD 600 value could reach 98 when dissolved oxygen was controlled at 50% and glucose around 1%.The production of human-like collagen with a yield of 29.4% was obtained.