Developing a potential prebiotic of yogurt: growth of Bifidobacterium and yogurt cultures with addition of glycomacropeptide hydrolysate

This study is aimed at investigating the feasibility of casein glycomacropeptide (GMP) hydrolysates as potential prebiotics in yogurt. The growth performance of Bifidobacterium animalis subsp. lactis (Bb12) was determined in the presence of GMP hydrolysates produced by the action of proteolytic enzymes (trypsin and papain) with various degrees of hydrolysis. The growth‐promoting effect of GHP on Lactobacillus bulgaricus and Streptococcu thermophilus were also evaluated. Results showed that the optimum hydrolysis time of GMP with trypsin or papain for Bb12 growth promotion was 2 and 0.5 h, respectively. Compared with GMP and its trypsin hydrolysate, the GMP hydrolysate produced with papain (GHP) was the best for Bb12 growth, which obtained the highest viable count (9.3 log cfu mL^?1) and the lowest pH of 4.69. The obtained GHP significantly improved the growth of S. thermophilus (P < 0.05), while it had little effect on the growth of L. bulgaricus (P > 0.05)., The viable count of Bb12 of the yogurt obtained with the addition of 1.5% GHP was about four times higher than that of the control without GHP addition. The growth‐promoting effect of GHP might be related to its high content of Glu, Leu and Ala while had no direct relationship with sialic acid content.     

Physicochemical and bitterness properties of enzymatic pea protein hydrolysates

ABSTRACT:  The effects of different proteolytic treatments on the physiochemical and bitterness properties of pea protein hydrolysates were investigated. A commercial pea protein isolate was digested using each of 5 different proteases to produce protein hydrolysates with varying properties. After 4 h of enzyme digestion, samples were clarified by centrifugation followed by desalting of the supernatant with a 1000 Da membrane; the retentates were then freeze-dried. Alcalase and Flavourzyme™ produced protein hydrolysates with significantly higher ( P < 0.05) degree of hydrolysis when compared to the other proteases. Flavourzyme, papain, and alcalase produced hydrolysates that contained the highest levels of aromatic amino acids, while trypsin hydrolysate had the highest levels of lysine and arginine. Papain hydrolysate contained high molecular weight peptides (10 to 178 kDa) while hydrolysates from the other 4 proteases contained predominantly low molecular weight peptides (≤ 23 kDa). DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of the Flavourzyme hydrolysate was significantly ( P < 0.05) the highest while alcalase and trypsin hydrolysates were the lowest. Inhibition of angiotensin converting enzyme (ACE) activity was significantly higher ( P < 0.05) for papain hydrolysate while Flavourzyme hydrolysate had the least inhibitory activity. Sensory analysis showed that the alcalase hydrolysate was the most bitter while papain and α-chymotrypsin hydrolysates were the least. Among the 5 enzymes used in this study, papain and α-chymotrypsin appear to be the most desirable for producing high quality pea protein hydrolysates because of the low bitterness scores combined with a high level of angiotensin converting enzyme inhibition and moderate free radical scavenging activity.     

Generation,Fractionation, and Characterization of Iron‐Chelating Protein Hydrolysate from Palm Kernel Cake Proteins

Palm kernel cake protein was hydrolyzed with different proteases namely papain, bromelain, subtilisin, flavourzyme, trypsin, chymotrypsin, and pepsin to generate different protein hydrolysates. Peptide content and iron‐chelating activity of each hydrolysate were evaluated using O‐phthaldialdehyde‐based spectrophotometric method and ferrozine‐based colorimetric assay, respectively. The results revealed a positive correlation between peptide contents and iron‐chelating activities of the protein hydrolysates. Protein hydrolysate generated by papain exhibited the highest peptide content of 10.5 mM and highest iron‐chelating activity of 64.8% compared with the other hydrolysates. Profiling of the papain‐generated hydrolysate by reverse phase high performance liquid chromatography fractionation indicated a direct association between peptide content and iron‐chelating activity in most of the fractions. Further fractionation using isoelectric focusing also revealed that protein hydrolysate with basic and neutral isoelectric point (pI) had the highest iron‐chelating activity, although a few fractions in the acidic range also exhibited good metal chelating potential. After identification and synthesis of papain‐generated peptides, GGIF and YLLLK showed among the highest iron‐chelating activities of 56% and 53%, whereas their IC₅₀ were 1.4 and 0.2 μM, respectively.     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

Milk-based Gels Made with κ-Carrageenan

ABSTRACT: The gel strength of κ‐carrageenan (0.1 to 0.4% w/w) ‐ reconstituted skim milk (2.5 to 20% w/w milk solids) mixtures was influenced by the concentrations of milk solids, κ‐carrageenan and cations. Particle size measurements showed that particle interactions in diluted skim milk‐κ‐carrageenan mixtures were dependent on the conformation of the κ‐carrageenan. Heat treatment of milk, resulting in alteration of the casein micelle, did not affect the interaction of κ‐carrageenan with casein in dilute solutions or the gel strength of milk‐κ‐carrageenan mixtures. κ‐Carrageenan must be available in the random coil form in solution, prior to cooling to its coil to helix transition temperature, for effective gelation of milk‐κ‐carrageenan mixtures.     

Preparation and characteristics of yak casein hydrolysate–iron complex

The effect of mass ratio between yak casein hydrolysate and FeSO₄, reaction temperature, pH and holding time on ferrous‐binding capacity of yak casein hydrolysate was investigated. Iron‐releasing percentage and structural characteristics of the formed yak casein hydrolysate–iron complex were also examined. Results showed that casein hydrolysates with different hydrolysis degrees (DH) possessed different ferrous‐binding capacities with the highest of 7‐h hydrolysate. The optimal binding conditions between yak casein hydrolysate and iron are mass ratio of casein hydrolysate to FeSO₄ of 15:1 for 20 min at 40 °C and pH 6.0. The principal binding site for Fe²⁺ in yak casein hydrolysate consists of the carboxylate groups and amide groups. Compared with ferrous sulphate, yak casein hydrolysate–iron complex has higher iron‐releasing percentage at basic conditions, such as pH 7.0, 7.5 and 8.0. Ferrous‐iron enriched casein hydrolysate, i.e. yak casein hydrolysate–iron complex, may have great potential as an effective delivery vehicle of bioavailable iron.     

Hypocholesterolaemic effect of Bifidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate

Hypercholesterolaemia is one of the most important risk factors in the development of cardiovascular disease. In this study, we report the in vitro potential of Bifidobacterium animalis subsp. lactis (Bb12) cultures and bovine casein hydrolysates formed by trypsin and Bb12 culture to reduce cholesterols levels. Cholesterol levels in vitro were reduced by up to 48% after incubation with Bb12 and up to 87% after incubation with trypsin hydrolysates, whereas unhydrolysed bovine casein did not affect cholesterol levels. Individual peptide fractions, obtained from size-exclusion chromatography, from casein hydrolysates formed by trypsin after a 48 h hydrolysis, reduced cholesterol levels by 2.7–50%. The molecular masses of these fractions, containing hypocholesterolaemic peptides, were below 1200 Da, as determined by LC-MS.     

Debittering Mechanism in Bitter Peptides of Enzymatic Hydrolysates from Milk Casein by Aminopeptidase T

The bitter peptide fraction present in casein hydrolysates obtained by using three proteases (subtilisin, papain and trypsin) was treated with aminopeptidase T from Thermus aquaticus YT-1. The bitterness of the bitter peptide fraction could be decreased, and it sometimes disappeared completely, with an increase in free amino acids. The percentages of total free amino acids released from each bitter peptide fraction (subtilisin, papain and trypsin) by aminopeptidase digestion for 20 hr were approximately 11%, 8.7%, and 6.5%, respectively. Bitter peptide (α_s1-CN f91-100) was isolated from a tryptic hydrolysate of casein by HPLC, its threshold value of bitterness being 2.9 ppm (w/v). The peptide (α_s1-CN f96-100) obtained from the amino peptidase digestion of this bitter peptide showed no bitterness.     

Direct induction of CCK and GLP-1 release from murine endocrine cells by intact dietary proteins

Scope: Consumption of high‐protein diets cause elevated levels of CCK and GLP‐1. Although unknown, this might be due to protein breakdown by various proteases that originate from the gastrointestinal tract. This study investigated which dietary proteins, hydrolysates, or synthetic‐peptides are most potent to affect secretion of CCK and GLP‐1 in STC‐1 cells known for satiety hormone release. Methods and results: Addition of intact proteins to STC‐1 cells exerted strong effects on secretion of satiety hormones. Casein, whey, and pea showed strongest effects on CCK release, whereas casein, codfish, egg, and wheat showed most pronounced effects on GLP‐1 release. Egg‐hydrolysate stimulated release of CCK and GLP‐1, whereas all other tested hydrolysates and synthetic‐peptides showed no significant effects on hormone release. Addition of a combination of trypsin and casein‐hydrolysate, codfish, egg, egg‐hydrolysate, sodium‐casein, wheat‐hydrolysate, or wheat resulted in additional stimulation of CCK release, compared to only the protein. Addition of a combination of DPP‐IV and egg‐hydrolysate, ovomucoid, or sodium‐casein decreased GLP‐1 levels. Conclusion: This study showed that specific intact, or partially digested proteins, in contrast to protein‐hydrolysates and synthetic‐peptides, stimulated hormone release. We conclude that intact proteins exert strong effects on satiety hormone release, and may therefore provide potent dietary supplements for prevention or treatment of obesity.     

COMPARISON OF THE LYMPHOPROLIFERATION ACTIVITY OF YAK MILK CASEIN HYDROLYSATES HYDROLYZED BY MICROBIAL-DERIVED AND ANIMAL-DERIVED PROTEINASE

To select a kind of practical protease that can be used for the production of yak milk casein enzymatic hydrolysates with a high‐immunoregulating activity, the effects of casein trypsin, pepsin, alcalase hydrolysates and their separated fractions on the lymphoproliferation activity and Interleukin‐2 production of murine splenocytes in vitro were studied. Results showed that, compared with casein trypsin and pepsin hydrolysates, casein alcalase hydrolysate had higher immunoregulatory activity, degree of hydrolysis and peptide yield. The molecular weights of the separated fractions of casein hydrolysates were lower than 1,500 Da. Considering the low price and commercial availability in a large supply of alcalase, yak milk casein alcalase hydrolysate is more practical as a functional food ingredient in industry.     

Preparation and characterisation of an easily absorbabl Mg‐casein hydrolysate complex produced through enzymatic hydrolysis and ultrafiltration

In this study, we synthesised a Mg‐casein hydrolysate complex that allowed the effective absorption of Mg. The type of enzyme (papain, alcalase 2.4 L, pepsin, trypsin) and the enzyme/substrate ratio for casein hydrolysis was optimised. When the enzyme/substrate ratio was 30%, the alcalase 2.4 L‐hydrolysate showed the highest Mg‐chelation efficiency, of 96.1%. To characterise and enhance the function of casein hydrolysate, we fractionated the casein hydrolysate according to molecular weight using ultrafiltration. The Mg‐chelation efficiency was increased with the decrease in the molecular‐weight range of the hydrolysate fractions. The smallest casein hydrolysate (fraction 5, 1 kDa<) is used for preparation of Mg‐casein hydrolysate complex. Synthesised Mg‐casein hydrolysate complex (fraction 5) exhibited 100% Mg solubility and 39.5% Mg bioavailability. These results indicated that the Mg‐casein hydrolysate remained a stable chelate during simulated gastro‐intestinal digestion in vitro. The Mg‐casein hydrolysate complex exhibited excellent antioxidant activity as well as Mg binding.     

Protein hydrolysate from salmon frame debittered by plastein reaction: amino acid composition,characteristics and antioxidant activities

Impacts of plastein reaction on bitterness, physicochemical and antioxidant properties of salmon frame hydrolysate with the aid of various proteases (alcalase and papain) at different concentrations and varying reaction temperatures were investigated. Plastein was produced from hydrolysate by papain at 40°C, which had 30% degree of hydrolysis (30DHP). Rearrangement of peptides in hydrolysate was performed by 1% papain at 40°C for 10 h, yielding plastein namely ‘30DHP-P1’. It showed the lowest bitterness (P < 0.05) than other plasteins and hydrolysates. Surface hydrophobicity was not related well with bitterness. Therefore, the size of peptides also determines the bitterness. 30DHP-P1 had augmented solubility; however, its antioxidant activities (DPPH and ABTS radical scavenging activities and ferric reducing antioxidant power) were slightly lower (P < 0.05) than those of hydrolysates. Bitterness of hydrolysate was markedly debittered via plastein reaction under optimal condition. Plastein generally had lighter colour and still possessed antioxidant activity.     

Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin.

The antibacterial properties of enzymatic hydrolysates of bovine lactoferrin were examined to determine whether active peptides are produced from this protein. Hydrolysates prepared by cleavage of lactoferrin with porcine pepsin, cod pepsin, or acid protease from Penicillium duponti showed strong activity against Escherichia coli O111, whereas hydrolysates produced by trypsin, papain, or other neutral proteases were much less active. Low molecular weight peptides generated by porcine pepsin cleavage of lactoferrin showed broad-spectrum antibacterial activity, inhibiting the growth of a number of Gram-negative and Gram-positive species, including strains that were resistant to native lactoferrin. The antibacterial potency of the hydrolysate was at least eightfold greater than that of undigested lactoferrin with all strains tested. The active peptides retained their activity in the presence of added iron, unlike native lactoferrin. The effect of the hydrolysate was bactericidal as indicated by a rapid loss of viability of E. coli O111. The lactoferrin hydrolysate described in the present study has commercial value as a natural preservative agent for use in foods and cosmetics, and as a functional component of new clinical foods for prevention or treatment of gastrointestinal disease.     

Comparison of hydrolysis characteristics on defatted peanut meal proteins between a protease extract from Aspergillus oryzae and commercial proteases

A crude protease extract (CPE) was prepared from Aspergillus oryzae HN 3.042 in this work. Three commercial proteases (Alcalase 2.4L, Protamex and papain) and CPE were employed to hydrolyse defatted peanut meal (DPM). CPE was found to be the most effective protease with protein recovery of 80.6%. Moreover, CPE produced a higher degree of hydrolysis (DH, 43.4%) than the tested commercial proteases. The test of molecular weight distribution indicated that DPM proteins were mainly consisted of >10 KDa fraction (86.6%), whereas 3–6 KDa fraction was observed to be the main fraction of all the hydrolysates. CPE hydrolysate possessed a higher nutritional quality than DPM and other hydrolysates on the basis of FAO/WHO (1991) reference pattern. The sensory taste evaluation showed that CPE hydrolysate had better taste than other hydrolysates.     

Influence of whey peptides on the surface activity of κ‐casein and β‐lactoglobulin A

Whey protein hydrolysate (WPH) was fractionated by reverse‐phase chromatography to obtain fractions of varying surface‐hydrophobicities. A model oil–water interface (MI) was pre‐coated with the WPH or fractions thereof. Contact angle (θ) of sessile drops of κ‐casein (κ‐CN) or β‐lactoglobulin A (β‐LGA) were measured on the MI. Pre‐coating of MI with un‐fractionated WPH decreased θ, that is, increased surface activity, of both κ‐CN (35–8.3°) and β‐LGA (38–21.3°). Conversely, pre‐coating of MI with the fractions significantly increased θ of both proteins as a function of hydrophobicity. Data provide insight into variability of whey protein functionality in food applications.     

A Survey of the Peptide Profile in Prato Cheese as Measured by MALDI‐MS and Capillary Electrophoresis

In this study, we describe the characterization of the peptide profile in commercial Prato cheese by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and capillary electrophoresis (CE). Ten commercial Prato cheese brands were characterized via their physicochemical composition and subjected to fractionation according to solubility at pH 4.6. The pH 4.6 insoluble fraction was evaluated by CE, whereas MALDI‐MS was applied to the fraction soluble at pH 4.6 and in 70% ethanol. CE revealed a characteristic pattern of hydrolysis, with formation of para‐κ‐casein, hydrolysis of α_s1‐casein at the Phe₂₃ ‐ Phe₂₄ bond, and hydrolysis of β‐casein. For the MALDI‐MS data, a complex peptide profile was observed, with the identification of 44 peptides previously reported (24 peptides from α_s1‐casein, 14 from β‐casein, 3 from κ‐casein, and 3 from α_s2‐casein). It was also observed that cheeses with salt‐in‐moisture content greater than 5% showed an accumulation of a bitter‐tasting peptide (m/z 1536, α_s1‐CN f1‐13), suggesting a relationship between the higher salt concentration and the abundance of this peptide. In conclusion, the results showed that even commercial cheeses produced with different raw material and processing conditions showed very similar peptide profiles when assessed at the molecular level, and only 9 peptides were responsible for discrimination of cheeses.     

不同苦荞蛋白酶解产物抗氧化活性研究

以苦荞蛋白作为底物,采用碱性蛋白酶Alcalase 2.4 L、木瓜蛋白酶、胃蛋白酶、胰蛋白酶以及胃蛋白酶加胰蛋白酶模拟体内蛋白消化,制备苦荞麦蛋白水解物。采用DPPH及ABTS~+·法比较不同的蛋白水解物与水解前苦荞蛋白的体外抗氧化活性。结果表明:不同蛋白酶水解产物水解度由高到低的顺序为:碱性蛋白酶胃蛋白酶~胰蛋白酶胃蛋白酶木瓜蛋白酶胰蛋白酶,其中碱性蛋白酶水解苦荞蛋白水解度达29.95%。苦荞蛋白本身具有一定的抗氧化能力,其中DPPH清除率及ABTS~+·清除率最高分别达71.91%及11.25%,但均显著低于阳性对照Vc。随着水解程度的增加,苦荞蛋白水解产物抗氧化能力逐渐增强。其中,以碱性蛋白酶酶解产物抗氧化活性最高,其DPPH清除率及ABTS~+·清除率最高分别为91.65%(0.5 mg/mL)及16.67%(1 mg/mL),均显著高于原苦荞蛋白。其中,碱性蛋白酶水解产物的DPPH自由基清除率在高浓度(0.5mg/mL)条件下,与阳性对照Vc持平。同时碱性蛋白酶酶解产物抗氧化性(DPPH清除率及ABTS~+·清除率)显著优于其他蛋白酶解产物。因此,苦荞麦蛋白采用碱性蛋白酶解制备苦荞水解产物可作为天然的抗氧化剂。     

Angiotensin I-converting enzyme inhibitory and hypocholesterolemic activities: Effects of protein hydrolysates prepared from Achatina fulica snail foot muscle

The angiotensin I-converting enzyme (ACE) inhibitory activity and hypocholesterolemic effect of Achatina fulica snail foot muscle protein hydrolysates (SFMPH) and its hydrolysates were studied. The SFMPHs were prepared at a temperature of 121°C for 60 min. To obtain the enzymatic hydrolysates, the SFMPHs were further hydrolysed with three proteases (papain, trypsin, or alcalase). Among all the hydrolysates, alcalase hydrolysate showed the highest degree of hydrolysis and was dominated by a small molecular size fraction (189–686 Da). The SFMPH treated by alcalase was effective in disintegrating intact cholesterol micelles. Furthermore, alcalase hydrolysate with a hydrolysis time of 60 min showed a strong ACE inhibitory activity in vitro with an IC₅₀ of 0.024 mg/mL. Therefore, alcalase hydrolysate may be a promising ingredient for the use in functional foods.     

蓝圆鲹蛋白酶解物的螯合矿物离子活性研究

以蓝圆鲹为原料,采用胰蛋白酶、木瓜蛋白酶及碱性蛋白酶对其进行水解,研究酶解产物与钙、亚铁、锌离子的螯合活性。结果表明:蓝圆鲹经胰蛋白酶水解,水解度为23.14%时其产物具有较高螯合铁、锌离子的活性,其螯合率分别高达96.63%和94.28%;蓝圆鲹经木瓜蛋白酶水解,水解度为22.00%时,其产物具有较高螯合钙离子的活性,其螯合率高达96.78%;同时研究表明上述两种酶解物还具有较好的抗氧化活性,其1,1-二苯基-2-三硝基苯肼半抑制浓度值分别为3.74 mg/m L和3.64 mg/m L。此外,氨基酸分析表明蓝圆鲹高螯合矿物离子活性的酶解物中天冬氨酸、谷氨酸、赖氨酸含量较高。     

酪蛋白水解物对乳酸菌的促生长作用

研究了酪蛋白的木瓜蛋白酶水解物对乳酸菌(嗜热链球菌∶保加利亚杆菌=1∶1)的促生长作用.通过比较不同水解度的酪蛋白水解物的增值作用,发现酪蛋白经木瓜蛋白酶水解8 h后的水解物具有最强的促进乳酸菌生长作用.本试验进一步考察了该水解物对酸乳乳酸菌代谢产物(乳酸和胞外多糖)的产量及活菌数变化的影响.结果表明木瓜蛋白酶酪蛋白水...