Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Yokenella Regensburgei Infection in an Immunocompetent Patient.

BACKGROUND: Yokenella regensburgei is a gram-negative, opportunistic pathogen that is ubiquitous in natural environments. It commonly affects immunocompromised patients. METHODS: The patient was a 71-year-old female with skin and soft tissue wound caused by trauma. The surgical debridement and abscess drainage were performed by surgeons. The specie of infected organism was identified by the D2Mini semi-automated system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). RESULTS: The result of bacterium culture showed gram-negative bacillus was moist, grey-white, semitransparent, and regular shape. Then the D2Mini semi-automated system and MALDI-TOF revealed that the colonies are Y. regensburgei. According to the result for drug sensitivity, antibiotic therapy was switched to cefoperazone/sulbac-tam and levofloxacin. CONCLUSIONS: Cooperation between clinical microbiology laboratories and surgeons is essential for the infected patient without typical symptoms. Agricultural activities and stab wounds may be related to Y. regensburgei infection.

Characterization of mung bean endogenous proteases and globulins and their effects on the production of mung bean protein.

Mung bean protein possesses several health benefits, and aqueous processing methods are used for its production. However, mung bean protein yields are different with different methods, which are actually different in conditions (e.g., pH, temperature, and time). Herein, liquid chromatography tandem mass spectrometry identified 28 endopeptidases and exopeptidases in mung bean protein extract, and the positions of 8S and 11S globulins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were confirmed in our experimental conditions. The SDS-PAGE, trichloroacetic acid-nitrogen solubility index, and free amino acid analysis revealed that (1) 8S globulins showed strong resistance to the endopeptidases (optimal at pH 5 and 50 °C) at pH 3-9, and 11S globulin exhibit strong resistance expect at pH 3-3.5; (2) the exopeptidases (optimal at pH 6 and 50 °C) preferred to liberate methionine and tryptophan. These proteases negatively affected protein yield, and short production time and low temperature were recommended.

Comparison of physicochemical properties and volatile flavor compounds of plant-based yoghurt and dairy yoghurt.

The aim of this study was to investigate and compare the physicochemical characteristics and volatile flavor compounds of three kinds of yoghurt made from reconstituted milk, soy drink and oat drink. The results showed that with the same fermentation ending pH of 4.5, reconstituted yoghurt had the highest titratable acidity mainly due to the highest buffering capacity and microbial counts (LAB). The textural and water holding capacity (WHC) parameters revealed that soy-based yoghurt had the highest firmness, consistency and WHC, indicating more rigid gel was formed. Meanwhile, rheological analysis showed soy-based yoghurt owned higher G' and G'' values and higher stability against external stress, demonstrating that more and stronger interactions between soy proteins were built during fermentation. The confocal laser scanning microscopy (CLSM) image witnessed that soy-based yoghurt had the densest and finest network, while oat-based yoghurt had much coarser and looser structure, which was consistent with the lowest firmness and G' value for oat-based yoghurt. In terms of color, reconstituted yoghurt was the lightest and oat-based yoghurt showed more reddish and yellowish. The main volatile flavor compounds in all yoghurts were ketones, while aldehydes contributed more in soy and oat yoghurt. PCA plot showed that volatile flavor compounds of reconstituted yoghurt and oat-based yoghurt were relatively similar, while soy-based yoghurt was much more different with high OAVs of hexanal, 1-octen-3-one, 1-octen-3-ol and 2-octenal. This study supplied a theoretical basis and an improvement direction for the better development of healthier plant-based yoghurt similar to dairy yoghurt.

Phenolic compounds in walnut pellicle improve walnut (Juglans regia L.) protein solubility under pH-shifting condition.

This study focused on the interaction of walnut protein with phenolic extracts of walnut pellicle (PEWP) under alkaline condition, leading to enhancement of protein solubility under neutral condition. First, the change of PEWP under alkaline condition was determined by RP-HPLC and mass spectrometry, and the results showed that most ellagitannins in PEWP could be retained under alkaline condition within 3 h. Interaction between PEWP and walnut protein under pH-shifting condition resulted in the remarkable increase of protein solubility (above 90%) at neutral pH. The results from SDS-PAGE and SEC showed that the improved solubility lied in the formation of large and soluble protein aggregates due to the covalent interaction among walnut protein and polyphenols. A significant change in tertiary structure of protein-phenolic complex was witnessed by fluorescence spectrum and near-UV circular dichroism. Meanwhile, walnut protein-polyphenol interaction led to a slight increase in ß-turn while a slight decrease in ß-sheet. Combined with amino acid composition, it could be illustrated that the covalent bonding for walnut protein with polyphenol mainly occurred at Lysine residues.

Comparison of aroma active compounds in cold- and hot-pressed walnut oil by comprehensive two-dimensional gas chromatography-olfactory-mass spectrometry and headspace-gas chromatography-ion mobility spectrometry.

Aroma composition of cold-pressed walnut oil (CWO) and hot-pressed walnut oil (HWO) was analyzed by comprehensive two-dimensional gas chromatography-olfactory-mass spectrometry (GC × GC-O-MS) and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 83 and 197 compounds were identified in the CWO and HWO, respectively; among these, 76 and 123 compounds were sniffed exclusively by GC × GC-O-MS, respectively. A total of 36 volatile compounds were detected by HS-GC-IMS, of which 10 in CWO and 32 in HWO. Based on of flavor dilution (FD) factors, odor-activity values (OAVs), and recombination and omission experiments, 1-octen-3-ol, cyclohexanol, and benzaldehyde were found to be the key aroma-active compounds in CWO, while 3-methylbutanal, (E,E)-2,4-nonadienal, nonanal, 1-octen-3-ol, 3-pentanol, 1-octanol, and furfural were the key aroma-active compounds in HWO. Moreover, Maillard reaction and lipid oxidation were found to play an important role in flavor formation in HWO. This study provides a guide to improve the quality of walnut oil based on aroma characteristics.

Growing of fungi on the stored low denatured defatted soybean meals and the hydrolysis of proteins and isoflavone glycosides by fungal enzymes.

Recently, more and more attention has been paid to the effects of fungal contamination and fungal enzymes secreted in raw grain on product quality. As the starting material of protein and active components, the quality of low denatured defatted soybean meals (LDSM) directly determines the qualities of subsequent products. In previous studies, we have revealed that infection with Aspergillus ochraceus protease causes significant hydrolysis of proteins. In this study, growing of fungi on the stored low denatured defatted soybean meals (LDSM) was analyzed by high-throughput sequencing and real-time PCR, which revealed that the abundance of Aspergillus increased significantly after storage. Twenty fungal proteases and 9 fungal glucosidases were found in stored LDSM and zymography showed that the proteases were of serine-type with some cysteine and aspartic activities. Proteolysis of the soybean storage proteins mainly occurred after the hydration of LDSM and the average molecular weight of soy proteins decreased from 57.9 kDa to 30.7 kDa after 60 min's of hydrolysis. Two-dimensional electrophoresis (2-DE) analysis found the polypeptide fragments from soybean 7S and 11S proteins with molecular weight around 10-25 kDa in the hydrated LDSM. Glycosylated isoflavones were hydrolyzed in both dry and hydrated stored LDSM which resulted in significant (p < 0.05) increase in the contents of isoflavone aglycones. This study suggested that fungi contamination be a new factor affecting the properties of LDSM derived soy protein products.

Low-calorie bulk sweeteners: Recent advances in physical benefits, applications, and bioproduction.

At present, with the continuous improvement of living standards, people are paying increasing attention to dietary nutrition and health. Low sugar and low energy consumption have become important dietary trends. In terms of sugar control, more and more countries have implemented sugar taxes in recent years. Hence, as the substitute for sugar, low-calorie sweeteners have been widely used in beverage, bakery, and confectionary industries. In general, low-calorie sweeteners consist of high-intensity and low-calorie bulk sweeteners (some rare sugars and sugar alcohols). In this review, recent advances and challenges in low-calorie bulk sweeteners are explored. Bioproduction of low-calorie bulk sweeteners has become the focus of many researches, because it has the potential to replace the current industrial scale production through chemical synthesis. A comprehensive summary of the physicochemical properties, physiological functions, applications, bioproduction, and regulation of typical low-calorie bulk sweeteners, such as D-allulose, D-tagatose, D-mannitol, sorbitol, and erythritol, is provided.

Characterization of proteases from Irpex lacteus grown on minimally denatured soybean meal.

BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.

Formation and characterization of solid fat mimetic based on pea protein isolate/polysaccharide emulsion gels.

The emulsion gels that can be used as solid fat replacers were produced with different polysaccharides (κ-carrageenan, κC; high-acyl gellan, HA; konjac glucomannanon, and KGM), pea protein isolate (PPI) and sunflower seed oil. The effect of polysaccharide concentration on the texture, rheological property, microstructure, and water holding capacity of the mixed emulsion gels were investigated. Rheological results showed that the presence of polysaccharides enhanced the hardness, storage modulus and resistance against deformation of emulsion gel, where PPI/κC system exhibited superior hardness with a similar level of pig back fat, due to the self-gelation behavior of κC. CLSM and SEM results showed that the presence of κC, HA, and KGM broke the uniform structure of gel network and formed irregular, threadlike, and oval shaped inclusions respectively, resulting in the broken and coalescence of oil droplets. The α-helix content of emulsion gels decreased, while ß-sheet, ß-turn and random coils slightly increased due to the unfolding of protein during gel formation. This study may offer a valuable strategy for the development of solid fat mimetic with the characteristics closing to the pig back fat.

Efficient Utilization of Fruit Peels for the Bioproduction of D-Allulose and D-Mannitol.

Currently, the demand for low-calorie sweeteners has grown dramatically because consumers are more mindful of their health than they used to be. Therefore, bioproduction of low-calorie sweeteners from low-cost raw materials becomes a hot spot. In this study, a two-stage strategy was established to efficiently utilize D-fructose from fruit and vegetable wastes. Firstly, ketose 3-epimerase was used to produce D-allulose from D-fructose of pear peels. Secondly, the residual D-fructose was converted to D-mannitol by the engineered strain co-expression of D-mannitol 2-dehydrogenase and formate dehydrogenase. Approximately 29.4% D-fructose of pear peels was converted to D-allulose. Subsequently, under optimal conditions (35 °C, pH 6.5, 1 mM Mn2+, 2 g/L dry cells), almost all the residual D-fructose was transformed into D-mannitol with a 93.5% conversion rate. Eventually, from 1 kg fresh pear peel, it could produce 10.8 g of D-allulose and 24.6 g of D-mannitol. This bioprocess strategy provides a vital method to biosynthesize high-value functional sugars from low-cost biomass.

Effects of heat treatments on the properties of soymilks and glucono- δ - Lactone induced tofu gels.

Optimal heat treatment of the soymilks is important to the production of tofu. In this study, soymilk with protein concentration of 40 mg/mL were heated at different temperatures for the fixed 50 s and were characterized by surface hydrophobicity, disulfide linked protein species determined by non-reducing SDS-PAGE and protein structural elements determined by the circular dichroism (CD). Tofu gels were prepared by acidifying the heated soymilks at 60 ℃ and 80 ℃ respectively and gelation time, gel mechanical properties as well as gel viscoelastic properties were determined by rheological analysis. The results showed that most soymilks' properties except surface hydrophobicity changed rapidly when heating temperature was higher than 80 ℃. Gelation time, storage modulus (G') at the end of acidifying and cooling processes as well as retardation time (λ) and recovery rate of tofu gels were affected by the heat treatments of soymilks. The distances between the standardized data describing heated soymilks and tofu gels respectively were calculated and compared. It was found that gelation time, G' and λ were most closely related to disulfide bond linked polymer, [CD]222 and surface hydrophobicity respectively. This study will provide useful information to the improvement of tofu processing.

Hydrolyzing behaviors of endogenous proteases on proteins in sesame milk and application for producing low-phytate sesame protein hydrolysate.

Endogenous proteases with high activity have been identified in sesame seeds. However, the hydrolyzing behaviors of endogenous proteases on proteins in sesame milk are not well understood. In this study, the endogenous proteases optimally hydrolyzed proteins at pH 4.5 and 50 °C for 6 h. Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry analyses revealed that endogenous proteases randomly cleaved the cleavable peptide bonds on 11S globulins, and amino acid analysis indicated that serine carboxypeptidases preferentially cleaved tryptophan, phenylalanine, methionine, tyrosine, and leucine. The hydrolyzed sesame milk was separated into cream, transparent skim, and precipitate fractions by centrifugation (3000g, 5 min). The major protein components in skim were 42% peptides (<1500 Da) and 35% free amino acids. The phytate in skim was greatly reduced by adjusting to neutral and alkaline pH. This study is meaningful at supplying a strategy for producing low-phyate sesame protein hydrolysate.

Raw walnut kernel: A natural source for dietary proteases and bioactive proteins.

Walnut kernels are health-promoting nuts, which are mainly attributed to polyunsaturated fatty acids, phenolics, and phytosterols. However, the information concerning benefits of walnut proteins are limited. In this study, endopeptidases, aminopeptidases, carboxypeptidases, superoxide dismutases, catalases, and phospholipases with respective relative abundance of 2.730, 1.728, 0.477, 3.148, 0.743, and 0.173‰ were identified by liquid chromatography tandem mass spectrometry. These endogenous proteases exhibited activity in a broad pH range of 2-6.5, and optimal at pH 4.5 and 50 °C. Aspartic endopeptidases were predominant endopeptidases, followed by cysteine ones. There were two types of aspartic endopeptidases, one (not inhibited by pepstatin A) exerted activity at pH 2-3 and the other (inhibited by pepstatin A) optimal at pH 4.5. Carboxypeptidases were optimal at pH 4.5, and aminopeptidases exerted activity at pH near 6.5. These endogenous proteases assisted the digestion of walnut proteins, and soaking, especially peeling, greatly improved the in vitro digestibility.

Formation Mechanism of Hexanal and (E)-2-Hexenal during Soybean [Glycine max (L.) Merr] Processing Based on the Subcellular and Molecular Levels.

Hexanal and (E)-2-hexenal in soymilk mainly form during the soaking and grinding of soybeans. In this study, freshly dehulled soybeans were soaked or ground in the presence or absence of different enzyme inhibitors. The results showed that (1) 1-palmitoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-stearoyl-2-linoleoyl-sn-3-phosphatidylcholine, 1-palmitoyl-2-linolenoyl-sn-3-phosphatidylcholine, and 1-stearoyl-2-linolenoyl-sn-3-phosphatidylcholine were preferentially acted upon by lipoxygenases (LOXs) and made predominant contributions to hexanal/(E)-2-hexenal formation. Phospholipase A2 (PLA2) is one of the key enzymes for hexanal/(E)-2-hexenal formation. (2) The ratio of net increase in hexanal/(E)-2-hexenal and net decrease in linoleic acid/linolenic acid was close to 100% during soaking, but it was only 60% during grinding. Only 13-hydroperoxy octadecad(tr)ienoic acid (13-HPOD/T) was formed for the membrane LOX, but both 13- and 9-hydroperoxy octadecad(tr)ienoic acid (9-HPOD/T) were produced for the cytoplasm LOX. Thus, only the membrane LOX was involved during soaking, while both membrane- and cytoplasm-bound LOXs worked during grinding. (3) Hydroperoxides and hexanal/(E)-2-hexenal during soybean grinding were studied. PC hydroperoxides formed almost instantly and reached a maximum in 10 s, while fatty acid hydroperoxides and hexanal/(E)-2-hexenal formed relatively slowly and reached a maximum in 50 s. The experimental data were fitted to the integrated form of the Michaelis-Menten equation, and Km, Vmax, and kcat for the LOX, PLA2, and hydroperoxide lyase were obtained, respectively.

Endopeptidases, exopeptidases, and glutamate decarboxylase in soybean water extract and their in vitro activity.

The proteolytic activity of some soybean endogenous proteases have been clarified in the previous studies, but the information concerning the roles of these proteases and some other unknown ones during soybean processing are scarce. Herein, 16 endopeptidases, 13 exopeptidases, 24 inhibitors (two serpin-ZX and one subtilisin inhibitor firstly identified), and one glutamate decarboxylase were identified in the soybean water extract by the liquid chromatography tandem mass spectrometry analysis. Amongst the identified endopeptidases, just the aspartic endopeptidases (optimal at pH 2.5-3 and 35-45 °C) showed the detectable proteolytic activity by the tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis and protease inhibitor assay analyses, whereas serine, cysteine, and metallo- endopeptidases (except P34 probable thiol protease) did not. Free amino acid analysis showed that the exopeptidases and glutamate decarboxylase were optimal at pH 6 and 45 °C, and by 6 h incubation, the free amino acids and γ-aminobutyric acid almost doubled.

Sesame water-soluble proteins fraction contains endopeptidases and exopeptidases with high activity: A natural source for plant proteases.

Recently, the interest in the plant proteases has greatly increased. However, only a few of proteases are isolated from the hugely produced oilseeds for the practical utilizations. In this study, the raw sesame milk prepared from peeled sesame seeds was separated into floating, skim, and precipitate fractions by centrifugation. The predominant aspartic endopeptidases and serine carboxypeptidases, which exerted high synergetic activity at pH 4.5-5 and 50-60 °C, were identified in the skim by the liquid chromatography tandem mass spectrometry, Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protease inhibitor assay, trichloroacetic acid-nitrogen soluble index (TCA-NSI), and free amino acid analyses. By incubating the mixture (protein content, 2%) of skim and precipitate at pH 4.5 and 50 °C for 6 h, the TCA-NSI and free amino acids achieved to 38.42% and 3148 mg/L, respectively. Moreover, these proteases efficiently degraded the proteins from soybean, peanut, and bovine milk.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein.

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Characterization of endogenous endopeptidases and exopeptidases and application for the limited hydrolysis of peanut proteins.

Research concerning the utilization of oilseed endogenous proteases is scarce. Herein, we investigated the peanut proteases and their effects on peanut proteins. Liquid chromatography tandem mass spectrometry analysis showed that peanut contained several endopeptidases and exopeptidases. Protease inhibitor assay and analysis of cleavage sites showed that the obvious proteolytic activity at pH 2-5 and 20-60 °C was from aspartic endopeptidases (optimal at pH 3) and one legumain (pH 4). The above endopeptidases destroyed five and six IgE-binding epitopes of Ara h 1 at pH 3 and 4, respectively. Ara h 1 (>95%) and arachin (50-60%) could be hydrolyzed to generate 10-20 kDa and <4 kDa peptides at pH 3, which was enhanced by the pH 3 â†’ 4 incubation. Further, the limited hydrolysis improved the gel-forming ability and in vitro digestibility (approximately 15%) of peanut proteins. Free amino acid analysis showed that the activity of exopeptidases was low at pH 2-5.

Insights into the antibacterial activity of cottonseed protein-derived peptide against Escherichia coli.

In the study, antibacterial peptides were separated and identified from cottonseed protein hydrolysates and the interactions between antibacterial peptides and Escherichia coli were further investigated. Firstly, by using a combined strategy of Amberlite CG-50 ion exchange chromatography and reversed-phase high-performance liquid chromatography, three peptides with antibacterial activity were purified and identified, including HHRRFSLY, KFMPT, and RRLFSDY. Interestingly, HHRRFSLY and RRLFSDY exhibited higher inhibition activity with the IC50 value of 0.26 mg mL-1 and 0.58 mg mL-1 (p < 0.05), respectively. Flow cytometry results showed that the incubation of antibacterial peptides with E. coli could cause damage to the integrity of the E. coli cell membrane. Transmission electron microscopy and scanning electron microscopy results revealed the damage caused to the bacterial cell surface and the leakage of cytoplasmic content by the antibacterial peptides. Molecular docking studies indicated that HHRRFSLY, KFMPT, and RRLFSDY have a good binding affinity to the active sites of the surface protein (OmpF) mainly through a hydrogen bond and salt bridge. The results here showed that the antibacterial peptides derived from cottonseed protein could be used as a good choice for functional foods or related drugs, and also shed light on further studies of antibacterial mechanism.