Effect of hydrocolloids on starch digestion: A review.

Rapidly digestible starch can increase postprandial blood sugar rapidly, which can be overcome by hydrocolloids. The paper aims to review the effect of hydrocolloids on starch digestion. Hydrocolloids used to reduce starch digestibility are mostly polysaccharides like xanthan gum, pectin, ß-glucan, and konjac glucomannan. Their effectiveness is related to their source and structure, mixing mode of hydrocolloid/starch, physical treatment, and starch processing. The mechanisms of hydrocolloid action include increased system viscosity, inhibition of enzymatic activity, and reduced starch accessibility to enzymes. Reduced starch accessibility to enzymes involves physical barrier and structural orderliness. In the future, physical treatments and intensity used for stabilizing hydrocolloid/starch complex, risks associated with different doses of hydrocolloids, and the development of related clinical trials should be focused on. Besides, investigating the effect of hydrocolloids on starch should be conducted in the context of practical commercial applications rather than limited to the laboratory level.

The substitution sites of hydroxyl and galloyl groups determine the inhibitory activity of human pancreatic α-amylase in twelve tea polyphenol monomers.

Tea polyphenols have been reported as potential α-amylase inhibitors. However, the quantitative structure-activity relationship (QSAR) between tea polyphenols and human pancreas α-amylase (HPA) is not well understood. Herein, the inhibitory effect of twelve tea polyphenol monomers on HPA was investigated in terms of inhibitory activity, as well as QSAR analysis and interaction mechanism. The results revealed that the HPA inhibitory activity of theaflavins (TFs), especially theaflavin-3'-gallate (TF-3'-G, IC50: 0.313 mg/mL), was much stronger than that of catechins (IC50: 18.387-458.932 mg/mL). The QSAR analysis demonstrated that the determinant for the inhibitory activity of HPA was not the number of hydroxyl and galloyl groups in tea polyphenol monomers, while the substitution sites of these groups potentially might play a more important role in modulating the inhibitory activity. The inhibition kinetics and molecular docking revealed that TF-3'-G as a mixed-type inhibitor had the lowest inhibition constant and bound to the active sites of HPA with the lowest binding energy (-7.74 kcal/mol). These findings could provide valuable insights into the structures-activity relationships between tea polyphenols and the HPA inhibitors.

Modulation of visceral pain by brain nuclei and brain circuits and the role of acupuncture: a narrative review.

Visceral pain is a complex and heterogeneous pain condition that is often associated with pain-related negative emotional states, including anxiety and depression, and can exert serious effects on a patient's physical and mental health. According to modeling stimulation protocols, the current animal models of visceral pain mainly include the mechanical dilatation model, the ischemic model, and the inflammatory model. Acupuncture can exert analgesic effects by integrating and interacting input signals from acupuncture points and the sites of pain in the central nervous system. The brain nuclei involved in regulating visceral pain mainly include the nucleus of the solitary tract, parabrachial nucleus (PBN), locus coeruleus (LC), rostral ventromedial medulla (RVM), anterior cingulate cortex (ACC), paraventricular nucleus (PVN), and the amygdala. The neural circuits involved are PBN-amygdala, LC-RVM, amygdala-insula, ACC-amygdala, claustrum-ACC, bed nucleus of the stria terminalis-PVN and the PVN-ventral lateral septum circuit. Signals generated by acupuncture can modulate the central structures and interconnected neural circuits of multiple brain regions, including the medulla oblongata, cerebral cortex, thalamus, and hypothalamus. This analgesic process also involves the participation of various neurotransmitters and/or receptors, such as 5-hydroxytryptamine, glutamate, and enkephalin. In addition, acupuncture can regulate visceral pain by influencing functional connections between different brain regions and regulating glucose metabolism. However, there are still some limitations in the research efforts focusing on the specific brain mechanisms associated with the effects of acupuncture on the alleviation of visceral pain. Further animal experiments and clinical studies are now needed to improve our understanding of this area.

Brain nuclei and neural circuits in neuropathic pain and brain modulation mechanisms of acupuncture: a review on animal-based experimental research.

Neuropathic pain (NP) is known to be associated with abnormal changes in specific brain regions, but the complex neural network behind it is vast and complex and lacks a systematic summary. With the help of various animal models of NP, a literature search on NP brain regions and circuits revealed that the related brain nuclei included the periaqueductal gray (PAG), lateral habenula (LHb), medial prefrontal cortex (mPFC), and anterior cingulate cortex (ACC); the related brain circuits included the PAG-LHb and mPFC-ACC. Moreover, acupuncture and injurious information can affect different brain regions and influence brain functions via multiple aspects to play an analgesic role and improve synaptic plasticity by regulating the morphology and structure of brain synapses and the expression of synapse-related proteins; maintain the balance of excitatory and inhibitory neurons by regulating the secretion of glutamate, γ-aminobutyric acid, 5-hydroxytryptamine, and other neurotransmitters and receptors in the brain tissues; inhibit the overactivation of glial cells and reduce the release of pro-inflammatory mediators such as interleukins to reduce neuroinflammation in brain regions; maintain homeostasis of glucose metabolism and regulate the metabolic connections in the brain; and play a role in analgesia through the mediation of signaling pathways and signal transduction molecules. These factors help to deepen the understanding of NP brain circuits and the brain mechanisms of acupuncture analgesia.

Effects of different gelatinization degrees of starch in potato flour on the quality of steamed bread.

The effect of four kinds of potato flour with different gelatinization degrees on the quality of steam bread was investigated in the present study. Results showed that medium-well flour (MWF) and potato flakes (PF) steamed bread, particularly MWF steamed bread, possessed the desired product properties liked by consumers. The MWF steamed bread had the highest appearance score (42.78) and total sensory evaluation score (81.60), and the PF steamed bread exhibited the highest specific volume (1.84 mL/g) and taste score (43.05). An increase in the degree of potato flour gelatinization led to an increase in dough gas retention coefficient from 80.20 mL/100 mL to 85.17 mL/100 mL and a more uniform and dense dough microstructure. During dough preparation, the increased gelatinization degree of potato flours enhanced the hydroscopicity competition between potato starch and gluten, resulting in a flocculent gluten network and increased potato starch volume during steaming. During steaming, steamed bread with higher gelatinization degree of potato flour formed a homogenous and dense starch gel-gluten double network, making them softer with more uniform gas cells and larger specific volume. Thus, this study provides a perspective of the effect of starch gelatinization on steamed bread quality.

Impact of celluloses and pectins restrictions on gluten development and water distribution in potato-wheat flour dough.

The addition of potato to wheat flour extends the nutritional values of bread. However, the adverse effects mediated by high dietary fiber in potato flour could affect the formation of gluten matrix. The water dynamics and distribution determined by the Low field nuclear magnetic resonance (LF-NMR) demonstrated a competitive water binding of dietary fiber, resulting in the partial dehydration and conformational changes of gluten protein complexes. Besides, the microstructure of the dough characterized by Scanning electron microscope (SEM) suggested that the insoluble cellulose could block the continuity of gluten from the spatial position, thereby negative affecting the mechanical properties of the dough. In our study, addition of cellulase and/or pectinase apparently mitigated the gluten aggregation and dehydration, contributing to the formation and the continuity of the three-dimensional gluten network. As a consequence, the specific volume of the bread was increased by 40.2%, and the hardness was reduced by 64.48%.

RNA Sequencing Analysis of Gene Expression by Electroacupuncture in Guinea Pig Gallstone Models.

BACKGROUND: Clinical studies have shown that electroacupuncture (EA) promotes gallbladder motility and alleviates gallstone. However, the mechanism underlying the effects of EA on gallstone is poorly understood. In this study, the mRNA transcriptome analysis was used to study the possible therapeutic targets of EA. METHODS: Hartley SPF guinea pigs were employed for the gallstone models. Illumina NovaSeq 6000 platform was used for the RNA sequencing of guinea pig gallbladders in the normal group (Normal), gallstone model group (Model), and EA-treated group (EA). Differently expressed genes (DEGs) were examined separately in Model vs. Normal and EA vs. Model. DEGs reversed by EA were selected by comparing the DEGs of Model vs. Normal and EA vs. Model. Biological functions were enriched by gene ontology (GO) analysis. The protein-protein interaction (PPI) network was analyzed. RESULTS: After 2 weeks of EA, 257 DEGs in Model vs. Normal and 1704 DEGs in EA vs. Model were identified. 94 DEGs reversed by EA were identified among these DEGs, including 28 reversed upregulated DEGs and 66 reversed downregulated DEGs. By PPI network analysis, 10 hub genes were found by Cytohubba plugin of Cytoscape. Quantitative real-time PCR (qRT-PCR) verified the changes. CONCLUSION: We identified a few GOs and genes that might play key roles in the treatment of gallstone. This study may help understand the therapeutic mechanism of EA for gallstone.

Rosmarinic Acid Decreases the Malignancy of Pancreatic Cancer Through Inhibiting Gli1 Signaling.

BACKGROUND: Rosmarinic acid (RA) has been shown to exert anti-tumor effects on various types of cancer. However, its roles in the treatment of pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanisms remain elusive. PURPOSE: The present study aimed to investigate the therapeutic effects of RA on PDAC as well as the underlying mechanisms. STUDY DESIGN: Evaluation of the effects of RA on PDAC malignancy both in vitro and in vivo. METHODS: Cell counting kit 8 (CCK8) assay, colony formation assay, 5-Ethynyl-2'-deoxyuridine (EDU) incorporation assay, cell cycle analysis, and apoptosis assay were conducted to assess the inhibitory effect of RA on PDAC cell proliferation. Meanwhile, western blotting and RT-qPCR assay were performed to detect the target gene expression at protein and mRNA levels, respectively. Moreover, the in vivo anti-tumor activities of RA were assayed in an xenograft mouse model of PDAC. RESULTS: RA dramatically down-regulated Gli1 and its downstream targets. Further studies showed that RA prevents the nuclear translocation of Gli1, while promoting the degradation of cytosolic Gli1 via the proteasome pathway. Moreover, we observed that RA induced G1/S cell cycle arrest and apoptosis in the PDAC cells through regulating the expression of P21, P27, CDK2, Cyclin E, Bax, and Bcl-2, it inhibited the PDAC cell migration and invasion via E-cadherin and MMP-9. Notably, Gli1 overexpression markedly reversed the above RA-induced effects on PDAC cells, whereas Gli1 knockdown enhanced the effects. Additionally, the in vivo assays demonstrated that RA suppresses the tumor growth of PDAC presumably by inhibiting Gli1. CONCLUSION: We provided evidence that RA restrained the nuclear translocation of Gli1 and facilitates Gli1 degradation via proteasome pathway, reducing the malignancy of PDAC cells. These findings implicated RA as a therapeutic agent for PDAC.

Combinatorial effect of fermentation and drying on the relationship between the structure and expansion properties of tapioca starch and potato starch.

Naturally fermented and sundried tapioca starch is reportedly the traditional material for polvilho azedo and the primary ingredients of gluten-free food items in Brazil. This study aimed to investigate starch structure and expansion rate, high rate is acclaimed in baking application, changes of tapioca starch and potato starch during combinatorial fermentation and drying. The rate of expansion changed from 3.37 mL/g in native tapioca starch to 3.71 mL/g in fermented oven-dried tapioca starch and 6.97 mL/g in fermented sun-dried tapioca starch, while potato starch sample displayed lesser expansion on all treatments. Rapid viscosity analysis, size-exclusion chromatography, X-ray diffraction (XRD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) and electron paramagnetic resonance spectrometry (EPR) were performed to determine the structure and investigate its relationship with the expansion rate. Fermentation attacked amorphous area and cleaved glycosidic bonds. Sunlight exposure facilitated complex interactions, and crosslinking increased the molecular weight distribution (MWD) in fermented sun-dried potato starch and led to depolymerization in tapioca starch. EPR revealed an initial free radical distribution in both starches, and our results show that intensity changes in tapioca starch are essential for a high expansion capacity.

Study on rapid drying and spoilage prevention of potato pulp using solid-state fermentation with Aspergillus aculeatus.

Effects of solid-state fermentation on rapid drying and spoilage prevention of potato pulp were evaluated. Pectin hydrolyzing and antibacterial ability of pectinase-secreting Aspergillus aculeatus and Bacillus subtilis were compared. A. aculeatus grew better in potato pulp, with highest pectinase yield of 342.71 ±â€¯5.09 U/mL and rapid pH reduction to 3.76 ±â€¯0.01. Next generation sequencing showed that the abundance of genera Candida and Enterobacter, which probably caused undesirable fermentation and spoilage, were significantly reduced after inoculation with A. aculeatus. In addition, fermentation with A. aculeatus significantly reduced water holding capacity from 16.63 ±â€¯0.36 g/g to 7.78 ±â€¯0.12 g/g, which resulted in lower viscosity and water binding capacity, and concomitantly significantly decreased moisture content from 76.05 ±â€¯0.24% to 12.95 ±â€¯0.19% after filtration and airflow drying. These results suggested that solid-state fermentation might be a promising technology for efficient processing and utilization of potato pulp.

Characterization of physicochemical properties of cellulose from potato pulp and their effects on enzymatic hydrolysis by cellulase.

In this work, cellulose was extracted from potato pulp, a low-efficiently utilized by-product from potato starch industry, by a combined alkali and sodium sulfite treatment. Its physicochemical properties were characterized and compared with cellulose from corn stalk. The yield and purity of cellulose from potato pulp were determined to be 24.74% and 81.34% respectively. Cellulose from potato pulp had more loosened and porous structure, lower crystallinity index and larger specific surface area (SSA) compared with cellulose from corn stalk. It also provided the highest accessibility to cellulase (5.7 mg protein/g glucan) and had the highest enzymatic digestibility (with glucose yield of 88.46%). Maximum adsorption capacity (Wmax) and equilibrium constant (K) were obtained by fitting the adsorption data with the Langmuir adsorption isotherm, suggesting that cellulose from potato pulp had the highest cellulase adsorption efficiency. The data further indicated that the presence of non-cellulosic components, especially for pectin, appeared to have a considerable effect on the hydrolysis by cellulase due to non-productive adsorption. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue Wuxi 214122, Jiangsu Province, People's Republic of China.

A Multicenter, Randomized, Controlled Trial of Electroacupuncture for Perimenopause Women with Mild-Moderate Depression.

OBJECTIVE: Up to 62% of perimenopausal women have depression symptoms. However, there is no efficacy treatment. The aim of this study is to compare the clinical efficacy and safety of EA therapy and escitalopram on perimenopause women with mild-moderate depressive symptom. METHOD: A multicenter, randomized, positive-controlled clinical trial was conducted at 6 hospitals in China. 242 perimenopause women with mild-moderate depressive symptom were recruited and randomly assigned to receive 36 sessions of EA treatment or escitalopram treatment. The primary outcome measure was the 17-item Hamilton Depression Rating Scale (HAMD-17). The secondary outcome measures include menopause-specific quality of life (MENQOL) and serum sexual hormones which include estrogen, follicle-stimulating hormone, and luteinizing hormone. RESULTS: 221 (91.3%) completed the study, including 116 in the EA group and 105 in the escitalopram group. The baseline levels of demographic and outcome measurements were similar in the two groups. In the intervention period, there was no difference between two groups. However, in the follow-up, both HAMD-17 and MENQOL were significantly decreased, and at week 24 the mean differences were -2.23 and -8.97, respectively. There were no significant differences in the change of serum sexual hormones between the two groups. No serious adverse events occurred. CONCLUSION: EA treatment is effective and safe in relieving depression symptom and improving the quality of life in the perimenopausal depression. Further research is needed to understand long-term efficacy and explore the mechanism of this intervention. This study is registered with ClinicalTrials.gov NCT02423694.

Enzyme assisted fermentation of potato pulp: An effective way to reduce water holding capacity and improve drying efficiency.

The effects of an enzyme-assisted fermentation technique on the water holding capacity (WHC) and drying characteristics of potato pulp were investigated. Potato pulp was fermented with Lactobacillus plantarum to increase the content of organic acids, reduce the pH of the pulp and assist in enhancing the enzymatic hydrolysis. Removal of pectin and cellulose reduced the WHC of potato pulp from 16.84 to 6.83 and 9.64 g/g, respectively. Drying characteristics were determined using the air-drying process, which indicated that pectinase-assisted fermentation could remove pectin from potato pulp and shorten the air-drying time from 240 to 140 min, while cellulase had an opposite effect on the drying rate. This result was also confirmed by low field nuclear magnetic resonance (LF-NMR), which showed more mobile water and less bonded water in pectin-removed potato pulp samples.

[Herb-partitioned moxibustion on navel for anovulatory infertility:a randomized controlled trial].

OBJECTIVE: To observe the efficacy differences between herb-partitioned moxibustion on navel and clomiphene for anovulatory infertility. METHODS: With double-blind double-dummy randomized control method, a total of 40 patients with anovulatory infertility were randomly divided into a moxibustion group and a clomiphene group, 20 cases in each one. Blinding was conducted on both patients and doctors. The patients in the moxibustion group were treated with herb-partitioned moxibustion on navel and oral administration of clomiphene placebo, while the patients in the clomiphene group were treated with placebo-partitioned moxibustion on navel and oral administration of clomiphene. The herb-partitioned moxibustion and placebo-partitioned moxibustion were given at the end of menstruation, 1.5 hours per treatment, once a week, and no treatment was given during menstruation. The oral administration of clomiphene and clomiphene placebo were given from 5 days into menstruation, 50 mg, once a day, for consecutive 5 days. One menstrual cycle was taken as one treatment course, and 3 treatment courses were conducted. After 3 treatment courses, the endometrial thickness (ET), maximum follicular diameter (MFD), ovulation rate (OR) and effective rate (ER) were evaluated between the two groups. RESULTS: (1) Compared before treatment, ET was significantly increased after treatment in the two groups (both P<0.05); after treatment, the ET in the moxibustion group was higher than that in the clomiphene group (P<0.05). (2) After treatment, MFD was significantly increased in the moxibustion group (P<0.05) and insignificantly increased in the clomiphene group (P>0.05); the MFD in the moxibustion group was higher than that in the clomiphene group (P<0.05). (3) The OR was 75.0% (15/20) and 65.0% (13/20) in the two groups respectively, which were not significantly different (P>0.05). (4) The total ER in the moxibustion group was 95.0% (19/20), which was superior to 70.0% (14/20) in the clomiphene group (P<0.05). CONCLUSIONS: The clinical efficacy of herb-partitioned moxibustion at navel on anovulatory infertility was superior to that of clomiphene, but their effects on OR was similar.

Characterisation of physicochemical and functional properties of soluble dietary fibre from potato pulp obtained by enzyme-assisted extraction.

In this study, enzyme-assisted methodology was applied with the aim to optimise the efficiency of soluble dietary fibre (SDF) extraction from potato pulp. The yields of SDF pretreated by cellulase, xylanase, and a cellulase/xylanase mixture were 31.9% (w/w), 25.7% (w/w), and 39.7% (w/w) respectively. When comparing single enzyme pretreatment with combined enzymatic treatment, we observed differences in properties related to molecular weight and viscosity. SDF obtained by xylanase pretreatment rendered better results for glucose dialysis retardation index (32.98%, w/w), amylase inhibitory effect (56.2%), and pancreatic lipase inhibitory activity (55.33%), while combined enzymatic pretreatment exhibited better effect in properties related to exposure of functional groups and specific surface area, including sodium cholate binding capacity (72.2%) and hydroxyl radical scavenging activity (87.57%). Our results indicated that enzymatic pretreatment, which could effectively hydrolyse cellulose and hemicellulose components, could not only benefit the yield of SDF, but also enhance its physiological and functional properties.

[Clinical observation of herb-partition moxibustion at navel for constipation with excess syndrome].

OBJECTIVE: To observe the effect differences between herb-partition moxibustion and starch-partition moxibustion at navel for constipation with excess syndrome. METHODS: Sixty patients were randomly assigned into an herb-partition moxibustion at navel group (herbal group) and a starch-partition moxibustion at navel group (starch group), 30 cases in each one. The acupoint was Shenque (CV 8) in the two groups. The treatment was given once a week and 4 times totally. Scores for constipation degree and quality of life (PAC-QOL) were observed before and after treatment and at follow-up; clinical effects were compared between the two groups as well. RESULTS: After treatment, the scores of constipation degree and QOL were lower than those before treatment in the herbal group (P<0.01,P<0.05), which were better than those of the starch group (P<0.01,P<0.05). Good effects were achieved for heat constipation (84.6%), qi constipation (77.8%) and cold constipation (87.5%) in the herbal group, and the improvements of heat constipation and qi constipation were better than corresponding results in the starch group (both P<0.05). The effective rate of the herbal group was 83.3% (25/30), and it was better than 43.3% (13/30) of the starch group. The two scores at follow-up had no statistical significances compared with those after treatment in the herbal group (both P>0.05), but superior to ones of the starch group (P<0.01,P<0.05); the effective rate of the herbal group was 86.6% (26/30), which was better than 50.0% (15/30) of the starch group (P<0.01). CONCLUSIONS: Herb-partition moxibustion at navel achieves better short-term and long-term effects than starch-partition for constipation with excess syndrome.

A clinical trial of acupuncture about time-varying treatment and points selection in primary dysmenorrhea.

OBJECTIVE: To observe the clinical therapeutic effects of acupuncture at single point Shiqizhui (EX-B8) and multi-points in time-varying treatment for primary dysmenorrhea. METHODS: 600 patients with primary dysmenorrhea were randomly assigned to the single point group (n=200) including group A (treating before the menstruation, n=100) and group B (immediately treating as soon as pain occurrence, n=100), the multi-points group (n=200) including group C (treating before the menstruation, n=100) and group D (immediately treating as soon as pain occurrence, n=100), or the control group, group E (n=200, no treatment). The therapeutic effects were analyzed after treatment for three menstrual cycles and interviewed for three follow-up periods. RESULTS: Acupuncture could effectively relieve menstrual pain for primary dysmenorrhea compared with the control group (P<0.05, P<0.01). Immediate pain relief occurred following acupuncture within 5 min in group B (P<0.01) and group D (P<0.01), and the two groups obviously relieved menstrual pain for VAS scores. Both group A and group C obviously relieved menstrual pain (P<0.01), and group C was better than group A (P<0.05). Compared with group D, Group C was much better for CMSS scores in cycle 1. CONCLUSION: Treating before the menstruation is better than immediately treating as soon as pain occurrence at the improvement in symptoms of dysmenorrheal at multi-points. And single point is better than multi-points when immediately treating as soon as pain occurrence. The present trial suggest Shiqizhui (EX-B8) should be chosen as a convenient point.

Randomized clinical trial: the clinical effects of herb-partitioned moxibustion in patients with diarrhoea-predominant irritable bowel syndrome.

Objective. To explore the efficacy of Herb-partitioned moxibustion in treating IBS-D patients. Method. 210 IBS-D patients were randomly assigned on a 3 : 3 : 2 basis to group HM, group FM, or group PB for 4-week treatment. The change of GSRS total score at weeks 4 and 8, the changes of GSRS specific scores, and adverse events were evaluated. Results. Patients in group HM and group FM had lower GSRS total score at week 4 (1.98 ± 0.303, 2.93 ± 0.302 versus 3.73 ± 0.449) and at week 8 (2.75 ± 0.306, 3.56 ± 0.329 versus 4.39 ± 2.48) as compared with patients' score in group PB. However, there was no significant difference of GSRS total score between group HM and group FM. The effect of HM was significantly greater than that of orally taking PB in ameliorating the symptoms of rugitus (0.38 versus 0.59, P < 0.05), abdominal pain (0.28 versus 0.57, P < 0.01), abdominal distension (0.4 versus 0.7, P < 0.01), and increased passage of stools (0.06 versus 0.25, P < 0.01) at the end of treatment period. In the follow-up period, patients' therapeutic effect in group HM remained greater than that in group FM (in abdominal pain, abdominal distension, and increased passage of stools) and that in group PB (in loose stools). Conclusions. HM appears to be a promising, efficacious, and well-tolerated treatment for patients with IBS-D.

Delayed supplementation of glycine enhances extracellular secretion of the recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli.

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant alpha-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the alpha-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.

Calcium leads to further increase in glycine-enhanced extracellular secretion of recombinant alpha-cyclodextrin glycosyltransferase in Escherichia coli.

Overexpression of recombinant genes in Escherichia coli and targeting recombinant proteins to the culture medium are highly desirable for the production of industrial enzymes. However, a major barrier is inadequate secretion of recombinant protein across the two membranes of E. coli cells. In the present study, we have attempted to circumvent this secretion problem of the recombinant alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine, as a medium supplement, could enhance the extracellular secretion of recombinant alpha-CGTase in E. coli. In the culture with glycine at the optimal concentration of 150 mM, the alpha-CGTase activity in the culture medium reached 23.5 U/mL at 40 h of culture, which was 11-fold higher than that of the culture in regular TB medium. A 2.3-fold increase in the maximum extracellular productivity of recombinant alpha-CGTase was also observed. However, further analysis indicated that glycine supplementation exerted impaired cell growth as demonstrated by reduced cell number and viability, increased cell lysis, and damaged cell morphology, which prevented further improvement in overall enzyme productivity. Significantly, Ca(2+) could remedy cell growth inhibition induced by glycine, thereby leading to further increase in the glycine-enhanced extracellular secretion of recombinant alpha-CGTase. In the culture with 150 mM glycine and 20 mM Ca(2+), both extracellular activity and maximum productivity of recombinant enzyme were 1.5-fold higher than those in the culture with glycine alone. To the best of our knowledge, this is the first article about the synergistic promoting effects of glycine and Ca(2+) on the extracellular secretion of a recombinant protein in E. coli.