Isolation of a surfactin-producing strain of Bacillus subtilis and evaluation of the probiotic potential and antioxidant activity of surfactin from fermented soybean meal.

BACKGROUND: Surfactin, usually produced by microbial metabolism, has many advantages including low toxicity, high biodegradability, and stability at extreme pH levels and temperatures, making it suitable for industry. However, its commercial production has not yet been achieved. RESULTS: A strain with a strong surfactin-producing ability was isolated and identified as Bacillus subtilis SOPC5, based on the appearance of colonies, microscopic observation, and 16S rDNA sequencing. The isolate exhibited significant tolerance to acid, bile, gastric, and intestinal juices, and was sufficiently susceptible to antibiotics. Bacillus subtilis SOPC5 showed high levels of auto-aggregation and surface hydrophobicity, and a strong capacity to secrete protease, amylase, and cellulase. The strain also exhibited antibacterial activity against Staphylococcus aureus 10 306 with a antibacterial circle diameter of 18.0 ± 1.1 mm. The maximal yield of surfactin (1.32 mg mL-1) was obtained by fermenting soybean meal (SBM) using the isolate under the following conditions: SBM 86 g L-1, inoculation 1.5 × 107 CFU mL-1, FeSO4 1.2 mg L-1, MnSO4 2.6 mg L-1, MgSO4 0.5 mg mL-1, L-Glu 4 mg L-1, temperature 33 °C, duration 120 h, and shaking at 210 rpm. The purity of surfactin was 97.42% as measured by high-performance liquid chromatography (HPLC). The half inhibitory concentration (IC50) values for surfactin to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS·+) were 1.275 ± 0.11 and 0.73 ± 0.08 mg mL-1, respectively. CONCLUSION: This study provides a scientific basis for the application of B. subtilis SOPC5 (as a potential probiotic) and the preparation of its metabolic product (surfactin). © 2024 Society of Chemical Industry.

Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH1.5 shift, pH12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH 12 shift treatment. In addition, pH1.5 shift modified BSFLA tended to form ß-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH12-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.

ErbB3 is required for hyperaminoacidemia-induced pancreatic α cell hyperplasia.

Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver-α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mechanistic target of rapamycin complex 1, another ErbB3 downstream effector signal transducer and activator of transcription 3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mechanistic target of rapamycin complex 1 and signal transducer and activator of transcription 3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.

Nomogram Predicting Grade ≥2 Acute Radiation Enteritis in Patients With Cervical Cancer Receiving Concurrent Chemoradiotherapy.

OBJECTIVE: To analyze the risk factors for grade ≥2 ARE in patients with cervical cancer receiving concurrent chemoradiotherapy. METHODS: A total of 273 patients with cervical cancer receiving concurrent chemoradiotherapy at our hospital were retrospectively enrolled. The patients were divided into training and validation groups. Clinical parameters were analyzed using univariate analysis and multivariate logistic regression analysis. A nomogram model was established based on the independent risk factors selected using multivariate logistic regression. The areas under the receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis (DCA) were used to evaluate the nomogram. The patients were divided into low-score and high-score groups based on the scores calculated using the nomogram model and compared. RESULTS: Malnutrition, monocyte-lymphocyte ratio ≥0.82 after radiotherapy, platelet-lymphocyte ratio <307.50 after radiotherapy, and bowelbag volume receiving at least 5 and 40 Gy were independent risk factors for grade ≥2 ARE and were incorporated into the nomogram ( P <0.05). The ROC curve, calibration curve, and DCA suggested that the nomogram had good discrimination, concordance, and net benefit in the clinical. A medium nomogram score of 146.50 points was used as the cutoff point, and the incidence of grade ≥2 ARE in the high-score group was higher than that in the low-score group ( P <0.05). CONCLUSION: The nomogram model for grade ≥2 ARE has good predictive ability and clinical utility, and is convenient for clinicians to identify high-risk groups and develop early prevention and treatment strategies.

Application of fixed-frequency ultrasound in the cultivation of Saccharomyces cerevisiae for rice wine fermentation.

BACKGROUND: Rice wine (RW) fermentation is limited by its long fermentation time, weak taste and unpleasant flavors such as oil and odor. In this study, a novel ultrasound technology of Saccharomyces cerevisiae was used with the aim of improving fermentation efficiency and volatile flavor quality of RW. RESULTS: The results showed that fixed-frequency ultrasonic treatment (28 kHz, 45 W L-1, 20 min) of S. cerevisiae seed culture at its logarithmic metaphase significantly increased the biomass and alcohol yield by 31.58% and 26.45%, respectively, and reduced fermentation time by nearly 2 days. Flavor analysis indicated that the flavor compounds in RW, specifically the esters and alcohols, were also increased in quantity after the ultrasonic treatment of S. cerevisiae seed liquid. Isobutyl acetate, ethyl butyrate, ethyl hexanoate and phenethyl acetate contents were increased by 78.92%, 129.19%, 7.79% and 97.84%, respectively, as compared to the control. CONCLUSION: Ultrasonic treatment of S. cerevisiae reduced fermentation time and enhanced the flavor profile of RW. This study could provide a theoretical and/or technological basis for the research and development of RW. © 2024 Society of Chemical Industry.

Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein.

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

A GAPDH serotonylation system couples CD8+ T cell glycolytic metabolism to antitumor immunity.

Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.

Inhibition of cross-linked lysinoalanine formation in pH12.5-shifted silkworm pupa protein, and functionality thereof: Effect of ultrasonication and glycation.

We added three different carbohydrates (Xylose/Xyl, Maltose/Mal, and Sodium alginate/Sal) to pH12.5-shifted silkworm pupa protein isolates (SPPI), and examined the influence of multi-frequency ultrasound (US) on them, with reference to lysinoalanine (LAL) formation, changes in conformational characteristics and functionality. Results showed that, the LAL content of the glycoconjugates - SPPI-Xyl, SPPI-Mal, and SPPI-Sal decreased by 1.47, 1.39, and 1.54 times, respectively, compared with the control. Notably, ultrasonication further reduced the LAL content by 45.85 % and brought SPPI-Xyl highest graft degree (57.14 %). SPPI-Xyl and SPPI-Mal were polymerized by different non-covalent bonds, and SPPI-Sal were polymerized through ionic, hydrogen, and disulfide (covalent/non-covalent) bonds. Significant increase in turbidity, Maillard reaction products and the formation of new hydroxyl groups was detected in grafted SPPI (p < 0.05). US and glycation altered the structure and surface topography of SPPI, in which sugars with high molecular weight were more likely to aggregate with SPPI into enormous nanoparticles with high steric hindrance. Compared to control, the solubility at pH 7.0, emulsifying capacity and stability, and foaming capacity of SPPI-US-Xyl were respectively increased by 244.33 %, 86.5 %, 414.67 %, and 31.58 %. Thus, combined US and xylose-glycation could be an effective approach for minimizing LAL content and optimizing functionality of SPPI.

Disrupted RNA editing in beta cells mimics early-stage type 1 diabetes.

A major hypothesis for the etiology of type 1 diabetes (T1D) postulates initiation by viral infection, leading to double-stranded RNA (dsRNA)-mediated interferon response and inflammation; however, a causal virus has not been identified. Here, we use a mouse model, corroborated with human islet data, to demonstrate that endogenous dsRNA in beta cells can lead to a diabetogenic immune response, thus identifying a virus-independent mechanism for T1D initiation. We found that disruption of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) in beta cells triggers a massive interferon response, islet inflammation, and beta cell failure and destruction, with features bearing striking similarity to early-stage human T1D. Glycolysis via calcium enhances the interferon response, suggesting an actionable vicious cycle of inflammation and increased beta cell workload.

Effect of pectin concentration on emulsifying properties of black soldier fly (Hermetia illucens) larvae albumin modified by pH-shifting and ultrasonication.

The effect of pectin concentration on the structural and emulsifying properties of black soldier fly larvae albumin (BSFLA) modified by pH-shifting (pH12) and ultrasound (US) was studied. The results (intrinsic fluorescence, surface hydrophobicity, Fourier transform infrared spectrum, and disulfide bonds) showed that modified BSFLA samples, especially pH12-US, were more likely to bind to pectin through hydrogen bonding, electrostatic interactions, and hydrophobic interactions due to the unfolding of BSFLA, the collapse of disulfide bonds and exposure of hydrophobic groups. Thus, a BSFLA-pectin complex with smaller particle size, more negative charges, and a relatively loose structure was formed. The emulsifying activity (EAI) and stability index (ESI) of pH12-US modified BSFLA were significantly enhanced by the addition of pectin, reaching the highest values (associated with 174.41 % and 643.22 % increase, respectively) at pectin concentration of 1.0 %. Furthermore, the interface modulus of the emulsion prepared by the modified BSFLA was mainly viscous, and had higher apparent viscosity, smaller particle size and droplet size, contributing to higher EAI and ESI. The study findings suggest the addition of pectin to pH12-US treated BSFLA could be used in industry to prepare BSFLA-pectin emulsion with exceptional/desirable properties.

Preparation of housefly (Musca domestica) larvae protein hydrolysates: Influence of dual-sweeping-frequency ultrasound-assisted enzymatic hydrolysis on yield, antioxidative activity, functional and structural attributes.

Dual-sweeping-frequency ultrasound (DSFU) was utilized in the preparation of polypeptides from housefly (Musca domestica) larvae protein (HLP). Results indicated that ultrasonication (20 ± 2/28 ± 2 kHz, 42 W/L, 25 min) significantly increased peptide yield and DPPH scavenging capacity by 8.25 % and 14.83 %, respectively. Solubility, foaming and emulsification properties of polypeptides were improved by 19.89 %, 33.33 % and 38.74 % over the control; along with notable reduction in particle size and increase in zeta potential. Tertiary structural changes of the sonicated hydrolysates were illustrated by UV and fluorescence spectra. FTIR showed that ultrasonication increased α-helix, ß-turn, and random coil by 38.23 %, 46.35 % and 16.36 %, respectively, but decreased ß-sheet by 48.03 %, indicating partial unfolding in HLP hydrolysate conformation and reduction in intermolecular interactions. The research results demonstrated that dual-sweeping-frequency ultrasonication has a great prospect in industry application for the purpose of improving enzymolysis efficiency and product quality for housefly larvae protein hydrolysates production.

Hexagonal plate ultrasound pretreatment on the correlation between soy protein isolate structure and cholesterol-lowering activity of peptides, and protein's enzymolysis kinetics, thermodynamics.

In this study, a hexagonal plate ultrasound (HPU) pretreatment technology was employed to modify soy protein isolate (SPI) and enhance the hypocholesterolemic activity of enzymatic digests from SPI. Results demonstrated that under the condition of ultrasound power density of 40 W/L, the hypocholesterolemic activity of enzymatic digests from HPU-pretreated SPI (HPU-SPI) increased by 88.40 % compared to control group after gastrointestinal digestion. The sulfhydryl content of HPU-SPI increased by a maximum of 45.32 % compared to control group. Fourier transform infrared and scanning electron microscopy revealed that HPU pretreatment partially unfolded the SPI conformation, reduced the intermolecular interactions, and exposed the internal hydrophobic regions. Pearson correlation analysis showed that sulfhydryl groups (r = 0.860), disulfide bonds (r = -0.875) and random coil (r = 0.917) were strongly correlated with the cholesterol-lowering activity of soy protein hydrolysate (SPH), following a simulated gastrointestinal digestion. Finally, the effects of HPU pretreatment on enzymolysis kinetics and thermodynamics of the SPI enzymatic process showed that HPU pretreatment significantly reduced the Mie's constant, activation energy, activation enthalpy, activation entropy and Gibbs free energy. Overall, the study outcome suggested that HPU pretreatment could positively influence the hypocholesterolemic peptide activity, and thus, may be beneficial to the pharmaceutical/food industry.

Genetic risk converges on regulatory networks mediating early type 2 diabetes.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.

Nimotuzumab plus concurrent chemo-radiotherapy in unresectable locally advanced oesophageal squamous cell carcinoma (ESCC): interim analysis from a Phase 3 clinical trial.

BACKGROUND: This prospectively randomised, double-blinded, placebo-controlled, multicenter Phase 3 clinical trial was conducted to assess the efficacy and safety profile of nimotuzumab (nimo) plus concurrent chemo-radiotherapy (CCRT) in patients with unresectable locally advanced ESCC. METHODS: Patients were randomly assigned (1:1) to receive CCRT plus nimotuzumab or placebo. The primary endpoint was overall survival (OS). In addition, interim analysis for short-term response rate was pre-defined. RESULTS: A total of 201 patients were randomised into two groups. Eighty patients in the nimo group and eighty-two in the placebo group were evaluable. Three to six months after treatment, 26 (32.5%) patients achieved complete response (CR) in the nimo group, and 10 (12.2%) in the placebo group (P = 0.002). The ORR of the nimo group was significantly higher than the placebo group (93.8% vs. 72.0%, P < 0.001). The two groups' grade 3-5 adverse drug reactions were 11.1% vs. 10.9% (P > 0.05). CONCLUSIONS: Nimotuzumab, in combination with chemo-radiotherapy, increased the CRR and ORR with a good safety profile. The OS is needed to be followed and finally analysed. CLINICAL TRIAL REGISTRATION: NCT02409186.

[Retracted] miR­25 promotes metastasis via targeting FBXW7 in esophageal squamous cell carcinoma.

Following the publication of the above paper, it was drawn to the Editor's attention by a concerned reader that a large number of data panels showing cell migration and invasion assay data in Figs. 3C and 5 contained overlapping sections, such that data that were intended to show results obtained under different experimental conditions may have been derived from a smaller number of original sources. In addition, certain of the data in this pair of figures were strikingly similar to data that were submitted for publication in another journal at around the same time as the above paper was submitted to Oncology Reports. Finally, regarding the western blotting data shown in Fig. 4B, an obvious splice in the gel strip was noticed for the FBXW7 bands, whereas no equivalent splice was present in the associated GADPH loading control, suggesting that these data originated from different gels. Owing to the fact that the contentious data in the above article were under consideration for publication at around the time that this was submitted to Oncology Reports, in addition the other features of concern regarding the data, the Editor has decided that this paper should be retracted from the Journal on account of a lack of confidence in the presented data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 38: 3030­3038, 2017; DOI: 10.3892/or.2017.5995].

Enhancing the growth of thermophilic Bacillus licheniformis YYC4 by low-intensity fixed-frequency continuous ultrasound.

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca2+ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.

Modification of whey protein isolate by ultrasound-assisted pH shift for complexation with carboxymethylcellulose: Structure and interfacial properties.

The application of whey protein isolate (WPI) is limited because of its compact spherical structure. In this study, ultrasound-assisted pH shift was employed to modify WPI for complexation with carboxymethylcellulose (CMC). The foaming and emulsifying properties of WPI/CMC complexes were investigated. The results demonstrate that the pretreatment of ultrasound-assisted pH 12 shift increased the content of free sulfhydryl groups from 16.5 µmol/g to 34.7 µmol/g and enhanced protein hydrophobicity from 311.4 to 370.6 (p < 0.05). Compared to the complexes formed by untreated WPI and CMC, the complexes pretreated with ultrasound-assisted pH 12 shift had a smaller size of 293.4 nm and a more uniform distribution. Furthermore, WPI/CMC complexes pretreated by ultrasound-assisted pH 12 shift exhibited higher emulsifying activity and emulsion stability index, which were increased by 8.9 % and 42.6 % respectively, in comparison with the control group (p < 0.05). A positive correlation was found between the surface hydrophobicity of WPI and emulsifying activity of WPI/CMC complexes. Ultrasound-assisted pH 2 shift improved the foaming capacity of complexes by 28.3 % over the control (p < 0.05). All the results indicate that the interfacial properties of WPI/CMC complexes can be improved significantly by the combination of pH shift and ultrasound.

Pancreatic islet α cell function and proliferation requires the arginine transporter SLC7A2.

Interrupting glucagon signaling decreases gluconeogenesis and the fractional extraction of amino acids by liver from blood resulting in lower glycemia. The resulting hyperaminoacidemia stimulates α cell proliferation and glucagon secretion via a liver-α cell axis. We hypothesized that α cells detect and respond to circulating amino acids levels via a unique amino acid transporter repertoire. We found that Slc7a2ISLC7A2 is the most highly expressed cationic amino acid transporter in α cells with its expression being three-fold greater in α than ß cells in both mouse and human. Employing cell culture, zebrafish, and knockout mouse models, we found that the cationic amino acid arginine and SLC7A2 are required for α cell proliferation in response to interrupted glucagon signaling. Ex vivo and in vivo assessment of islet function in Slc7a2-/- mice showed decreased arginine-stimulated glucagon and insulin secretion. We found that arginine activation of mTOR signaling and induction of the glutamine transporter SLC38A5 was dependent on SLC7A2, showing that both's role in α cell proliferation is dependent on arginine transport and SLC7A2. Finally, we identified single nucleotide polymorphisms in SLC7A2 associated with HbA1c. Together, these data indicate a central role for SLC7A2 in amino acid-stimulated α cell proliferation and islet hormone secretion.

Synergistic effects of pH shift and heat treatment on solubility, physicochemical and structural properties, and lysinoalanine formation in silkworm pupa protein isolates.

The application of silkworm pupa protein isolates (SPPI) in food industry was limited because SPPI's solubility is poor and it contains a potential harmful component of lysinoalanine (LAL) which formed during protein extraction. In this study, combined treatments of pH shift and heating were performed to improve the solubility of SPPI and to reduce the content of LAL. The experimental results showed that the promoting effect on SPPI's solubility by alkaline pH shift + heat treatment was greater than that by acidic pH shift + heat. And an 8.62 times increase of solubility was observed after pH 12.5 + 80 â„ƒ treatment compared to the control SPPI sample which was extracted at pH 9.0 without pH shift treatment. Very strong positive correlation was found between alkali dosage and SPPI solubility (Pearson's correlation coefficient r = 0.938). SPPI with pH 12.5 shift treatment showed the highest thermal stability. Alkaline pH shift combined with heat treatment altered the micromorphology of SPPI and destroyed the disulfide bonds between macromolecular subunits (72 and 95 kDa), resulting in reduced particle size and increased zeta potential and free sulfhydryl content of the isolates. The fluorescence spectra analysis showed red shifts phenomena with pH increasing and fluorescence intensity increase with temperature increasing, implying the alterations in the tertiary structure of protein. Compared to the control SPPI sample, the amount of LAL reduced by 47.40 %, 50.36 % and 52.39 % using pH 12.5 + 70 â„ƒ, pH 12.5 + 80 â„ƒ and pH 12.5 + 90 â„ƒ treatment, respectively. These findings provide fundamental information for the development and application of SPPI in food industry.