Production and Characterization of ACE Inhibitory and Anti-Diabetic Peptides from Buffalo and Camel Milk Fermented with Lactobacillus and Yeast: A Comparative Analysis with In Vitro, In Silico, and Molecular Interaction Study

Abstract

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

Isolation, virtual screening, action mechanisms, chelation with zinc ions, and stability of ACE-inhibitory peptides from ginkgo seed globulin

“…4A), from which the IC 50 value of EASPKPV on inhibition capacity against ACE was calculated to be 87.66 ± 2.96 mmol L −1 . This value was lower than that of peptides YSCPHR identied from camel milk (IC 50 : 312 mmol L −1 ) and FMRWRD isolated from Tombul hazelnut (IC 50 : 259.7 mmol L −1 ), 24,34 but was higher than that of captopril, which was a widely used antihypertensive drug with a IC 50 of 0.14 mmol L −1 . 12 Since the IC 50 value was inversely proportional to the inhibiting power of peptides on ACE, EASPKPV showed a better inhibiting effect on ACE than YSCPHR and FMRWRD, but its inhibiting ability was poorer than that of captopril.…”

“…In the current study, ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC) were used in sequence to purify GSGH. 24 First, GSGH (2 mg mL −1 ) was pressed through an ultrafiltration membrane with an aperture of 0.22 μm (aqueous system, Jiteng Membrane Co., Tianjin, China). The percolate (2 mg mL −1 ) was loaded on a glass chromatographic column (16 × 1000 mm, Huwest, Xi'an, China), which was filled with Sephadex G-15 gel beforehand and equilibrated using dH 2 O.…”

Bitter Almond Albumin ACE-Inhibitory Peptides: Purification, Screening, and Characterization In Silico, Action Mechanisms, Antihypertensive Effect In Vivo, and Stability

“…Five oligopeptides, Gln-Pro-Pro-Ala-Ala-Ala-Ala-Ala-Ala-Gly-Ala-Gly (QPPAAAAAAGAG), Lys-Thr-Glu-Thr-Gln-Pro (KTETQP), Ser-Pro-Pro-Thr-Ala-Ala-Ala-Ala-Gly-Glu (SPPTAAAAGD), Arg-Pro-Pro-Ser-Glu-Asp-Glu-Asp-Gln-Glu (RPPSEDEDQE), and Thr-Cys-Gly-Ala-Ser (TCGAS) were obtained. However, only RPPSEDEDQE offered potential antihypertension based on the vector machine software score (1.35) calculated by the database AHTpin [ 28 ]. Thus, the sequence RPPSEDEDQE was chemically synthesized.…”

“…The BAAH (0.2 g/100 mL Milli-Q water) was first separated on a gel column (size: 8 × 100 cm, Xihu, Shanghai, China) chromatography filled with Sephadex G-25. The mobile phase was Milli-Q water with an elution speed of 3.2 mL every minute, and the monitored wavelength was 220 nm [ 28 ]. The corresponding peak fractions were collected, pooled, freeze-dried, and their inhibition ability against ACE was measured as per the N -hippuryl-L-histidyl-L-leucine method [ 30 ].…”

Biological Activities of Fermented Camel Milk Peptides: a Review

“…Camel milk is known as a distinct protein source, and after the degradation of its proteins, peptides are produced that have an inhibitory effect on the angiotensin-converting enzyme, which raises blood pressure, which is higher in fermented camel milk compared to other types of milk [20] . Angiotensinconverting enzymes stimulate the production of angiotensin II, which works to constrict blood vessels, and stimulates the release of the hormone aldosterone, which works to absorb sodium and thus regulate blood pressure [14].…”