Purification of goat beta-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor

This paper presents a novel contribution to the purification of goat beta-lactoglobulin by using an ultrafiltration membrane enzymic reactor. The basis of the purification process was the enzymic hydrolysis of contaminating proteins, alpha-lactalbumin and traces of serum albumin, by pepsin at 40 degrees C and pH 2, conditions under which beta-lactoglobulin is resistant to peptic digestion. Simultaneously, beta-lactoglobulin and peptides were separated by ultrafiltration. beta-Lactoglobulin was retained in the reactor while peptides generated by hydrolysis from alpha-lactalbumin and serum albumin permeated through the membrane. The process was made continuous by the addition of fresh whey to replace the lost permeate. Three mineral membranes with 10, 30 and 50 kDa molecular mass cut-off were tested and the 30 kDa membrane was selected for the continuous process. The simultaneous purification and concentration of beta-lactoglobulin from clarified goats' whey was achieved in a single step. The ultrafiltration membrane enzymic reactor could treat eight reactor volumes of clarified whey. The recovery of beta-lactoglobulin was 74%, its purity was 84% and its concentration 6.6-fold that in the initial clarified whey.     

Functional properties of soy protein hydrolysate produced from a continuous membrane reactor system

Two types of soy protein hydrolysates were produced by hydrolysis of isolated soy protein (ISP) with a combination of Alcalase and Flavourzyme in a continuous membrane system with 3000 and 30,000 daltons molecular-weight-cut-off (MWCO) membranes, respectively. Both hydrolysates were completely soluble over a pH range of 2–9. Their water-binding capacity increased 1.8–3.4 times at a water activity of 0.6–0.95 as compared to intact ISP. The antioxidant activity of ISP was remarkably enhanced by enzyme hydrolysis. The hydrolysate from the 30,000 daltons MWCO membrane had a much higher antioxidant activity than that from the 3000 daltons MWCO membrane. The results suggested that both selected enzymes and MWCO membrane governed the functional properties of protein hydrolysates. Long-term operation study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate over 16 h.     

乳清中棉籽蛋白的超滤回收研究

采用超滤技术回收蛋白质沉淀后的乳清中的棉籽蛋白,考察了操作压力、超滤时间、温度、pH、蛋白质浓度等因素对超滤膜通量的影响。结果表明,在等电点处膜通量最低,故超滤操作应在偏离蛋白质等电点的条件下进行;在实验范围内膜通量随操作压力的升高而升高,呈线性关系;随着超滤时间的延长,膜通量逐渐减小,超滤进行至20~30 min后,膜通量趋于稳定;随着温度的升高,膜通量增加。     

Antioxidant properties of protein hydrolysate from Douchi by membrane ultrafiltration

Protein extracted from Douchi was hydrolyzed by alcalase based on a single-factor experiment and response surface methodology was employed to optimize hydrolysis conditions. The optimum conditions were determined to be: 63℃ extraction temperature, 1.4% quantities of enzyme and substrate, and 1.7 h extraction time. The optimal protein hydrolysate was subsequently subjected to ultrafiltration, and the fraction of molecular weight 10–50 kDa was identified as the most potent contributor to antioxidant activity. Amino acid analysis indicated that acidic amino acids were the most abundant in all fractions of the protein hydrolysate. Infrared spectroscopy showed there were no significant differences in secondary structure between fractions.     

Angiotensin I-converting enzyme inhibitor derived from soy protein hydrolysate and produced by using membrane reactor

Five different proteolytic enzymes, including Alcalase, Flavourzyme, trypsin, chymotrypsin and pepsin were employed to hydrolyze isolated soy protein (ISP) to produce the hydrolysates, respectively. The result indicated that hydrolysis of ISP for 0.5–6 h with Alcalase produced the highest ACE inhibitory activity. Therefore, Alcalase was selected for further study on optimization of hydrolysis conditions. The optimum conditions for Alcalase to hydrolyze ISP to produce the lowest IC₅₀ value were: E/S = 0.01, hydrolysis temperature = 50 °C, pH 9.0 and hydrolysis time = 6 h. Under these conditions, the IC₅₀ value of ISP was significantly reduced from 66.4 to 0.67 mg protein/ml. The lower IC₅₀ value represented the higher the ACE inhibitory activity. Moreover, several membranes with molecular weight cut-offs (MWCFs) of 1000–30,000Da were used to filter the hydrolysate. The 10 kDa permeate obtained from the treatment of the hydrolysate by 10,000 Da MWCF membrane could further reduce its IC₅₀ value from 0.668 to 0.078 mg protein/ml with a peptide recovery of 67.5%. An operation stability study showed that the membrane reactor system could maintain a steady production of ISP hydrolysate for over 8 h. The in vitro effect of gastrointestinal protease on ACE inhibitory activity of 10 kDa permeate was also investigated. The results suggested that gastrointestinal proteases have very little effect on the ACE inhibitory activity of 10 kDa permeate.     

Production of an enzymic hydrolysate of casein on a kilogram scale

The scaling-up of a method for the production of an enzymic hydrolsate of casein is descrived; minor modifications of the laboratory procedure were required. The spray-dried product is relatively free of the bitterness often associated with proteins subjected to enzymic treatment. The bacteriological load of the raw materials, the intermediate stages, and the final product was monitored.
The production of hydrolysed casein in quantity is providing an opportunity to investigate the dietary potential of pre-digested protein for patients suffering from digestive disorders as a result of pancreatic malfunction.     

陶瓷膜超滤技术浓缩乳清的工艺参数研究

采用孔径为20nm的无机陶瓷膜超滤干酪副产物乳清,浓缩乳清蛋白。通过对膜过滤压力、温度以及乳清pH三个因素进行单因素分析以及正交实验优化,得到最佳工艺条件:操作压力0.25MPa,温度51℃,pH6.1,此条件下超滤膜渗透通量达到169.37L/m2.h,乳清蛋白可浓缩至5.4%,经喷雾干燥制得WPC蛋白质含量为38.2%。     

酶法生产乳清多肽饮料

采用碱性蛋白酶水解乳清多肽,制备具有较好热稳定性的可溶性多肽混合物,然后以其为母液,生产乳清多肽饮料。获得最优水解工艺的最适pH为9.0、最适温度为35℃,乳清中水解度高达25.4%。所研制的乳清多肽饮料无苦味,且具有良好的稳定性。     

Effect of pH and heat treatment of cheese whey on solubility and emulsifying properties of whey protein concentrate produced by ultrafiltration

The effects of pH and heat treatment of cheese whey on protein solubility (PS) at pH 4.6 and on emulsifying properties of whey protein concentrate (WPC) were studied by response surface methodology (RSM). PS followed a quadratic relationship with pH and a linear relationship with heat treatment. Highest values for PS were found between pH 6.0 and 6.6 with heat treatment at 68 °C for 2 min, and maximum solubility was reached at pH 6.3. Heat treatment strongly decreased protein solubility throughout the entire pH range. The emulsifying properties were notably benefited by raising the pH from 6 to 7, best values being shown at pH 7 with a heat treatment of 70 °C for 2 min. The effect of heat treatment on the emulsifying properties was dependent on the pH level. At about pH 7, the heat treatment adversely affected the emulsifying properties, probably due to excessive protein denaturation.     

Experimental evaluation of the allergen activity of a peptide preparation produced from an enzymatic hydrolysate of milk proteins using ultrafiltration]

Noninbred guinea-pig experiments were performed to investigate specific potential allergenicity of cow milk proteins (CMP) consisting of casein (80%) and serum proteins (20%), of low-molecular peptides (LMP 20/80) produced from CMP 20/80 by enzymatic hydrolysis and ultrafiltration; to evaluate nonspecific effect of test diet CMP 20/80 and LMP 20/80 on anaphylactic response of the animals to ovalbumin and histamine LD50. LMP 20/80 versus CMP 20/80 had a hypoallergenic effect. Potential mechanisms of low anaphylactic sensitivity induced by LMP 20/80 are discussed.     

Characterization of cold-set gels produced from heated emulsions stabilized by whey protein

This paper reports the cold gelation of preheated emulsions stabilized by whey protein, in contrast to, in previous reports, the cold gelation of emulsions formed with preheated whey protein polymers. Emulsions formed with different concentrations of whey protein isolate (WPI) and milk fat were heated at 90 °C for 30 min at low ionic strength and neutral pH. The stable preheated emulsions formed gels through acidification or the addition of CaCl₂ at room temperature. The storage modulus (G′) of the acid-induced gels increased with increasing preheat temperature, decreasing size of the emulsion droplets and increasing fat content. The adsorbed protein denatures and aggregates at the surface of the emulsion droplets during heat treatment, providing the initial step for subsequent formation of the cold-set emulsion gels, suggesting that these preheated emulsion droplets coated by whey protein constitute the structural units responsible for the three-dimensional gel network.     

Antinociceptive and anti-inflammatory effects of novel dietary protein hydrolysate produced from whey by proteases of Cynara cardunculus

Antinociceptive and anti-inflammatory effects of a peptide concentrate mix (PepC) obtained from whey protein, via hydrolysis with cardosins from Cynara cardunculus, was tested in vivo. The antinociceptive effect was assessed using writhing, hot-plate and formalin tests in mice, and the anti-inflammatory effect using the paw oedema test. PepC at 300 mg kg⁻¹ bw conveyed a significant result in the writhing test when co-administered with 1 and 3 mg kg⁻¹ bw indomethacin, similar to administration of higher doses of indomethacin alone. Conversely, no statistically significant differences were observed in the paw oedema test when the same PepC concentration was co-administered with dexamethasone at 3, 10 and 30 mg kg⁻¹ bw. PepC at 1000 mg kg⁻¹ bw did not cause any remarkable outcome in the hot-plate test. PepC appears to possess anti-inflammatory and peripheral antinociceptive activities, so it may be a candidate for nutraceutical ingredient.     

Characterization of major volatile compounds in whey spirits produced by different distillation stages of fermented lactose-supplemented whey

This research aimed to advance the understanding of acceptable sensory qualities of potable whey-based spirit from nonsupplemented, mid-supplemented, and high-supplemented whey samples by analyzing major volatile compounds during different stages of distillation (head, heart, and tail). The results demonstrated that commercial Saccharomyces cerevisiae strain in lactase-hydrolyzed whey showed rapid and complete sugar hydrolysis and efficient ethanol production in 24, 30, and 36 h on average, producing up to 29.5, 42.1, and 56.4 g/L of ethanol, respectively. The variations in titratable acidity, specific gravity, pH value, residual protein, sugar content, and alcohol yield were investigated during the fermentation. The total amount of volatile compound concentrations significantly decreased from the head (2,087–2,549 mg/L) to the tail whey spirits (890–1,407 mg/L). In the whey spirit, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, acetaldehyde, and ethyl acetate were the most prevalent dominant compounds, accounting for the largest proportion of total volatile compounds. The volatile compounds detected were far below the acceptable legal limit. The results suggest that high sensory qualities of potable whey-based spirits can be produced by fermentation of lactose-supplemented whey with S. cerevisiae cells.     

Characterisation of a whey protein hydrolysate as antioxidant

A whey protein hydrolysate was fractionated by size-exclusion chromatography to investigate the effectiveness of individual peptide fractions as (i) deactivators of hypervalent haeme iron, (ii) as radical scavengers, and (iii) as iron chelators. The reduction of ferrylmyoglobin, MbFe(IV)O, to metmyoglobin, MbFe(III), by the peptide fractions was found to follow first-order kinetics for excess peptide. The fractions with the highest rate of reduction of ferrylmyoglobin also exhibited most efficient radical scavenging as determined for the stable Fremy's salt radical. Inactivation of catalytic free ion by peptide fractions, as examined by their ability to inhibit formation of the ferrozine-Fe²⁺ complex, showed highest inhibition for the fractions with least scavenging capacity of the Fremy's salt radical. Radical scavenging of 1-hydroxylethyl radicals generated in Fenton reactions showed antioxidant activity for low peptide concentration, but prooxidative activity for increasing concentration. Specific whey peptide fractions, may be combined as a food additive to optimise antioxidative activity.     

Surfactant cleaning of ultrafiltration membranes fouled by whey

A polyethersulphone ultrafiltration membrane was prepared for concentration of whey. The membrane was fouled by whey and the effect of different cleaning agents on flux recovery of the fouled membrane was studied. The optimum cleaning procedure for membrane regeneration was elucidated. The results showed that a combination of surfactants (anionic, cationic and nonionic) may be employed as the optimum cleaning agent for maximum flux recovery. The fluorescence studies revealed that the cationic surfactant interact with proteins by breaking the intra‐chain hydrophobic bonding and providing electrostatic repulsion. Changing the alkyl chain from dodecyl to hexadecyl increases the interaction of surfactant–protein. Dodecyltrimethylammonium bromide (DTAB) provided a weak interaction with whey proteins than to tetradecyltrimethylammonium bromide (TTAB) and cetyltrimethylammonium bromide (CTAB). All data obtained in this study support a surfactant–protein interaction in which hydrophobic forces play a dominant role. The nonionic surfactants poly(oxyethylene) isooctyl phenyl ether (TX‐100) and anionic surfactants SDS interact with amino acids in the inner protein structure thus denaturate tertiary protein structure and reduce hydrophobic interaction of proteins by membrane surface.     

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.     

超滤膜分离技术回收乳清蛋白工艺研究

研究利用超滤膜分离技术,从干酪素乳清废弃液中回收乳清蛋白,通过对不同超滤膜性能的比较,选择最佳的超滤膜材料、工艺流程以及运行参数,并测得分离效果。结果表明:采用PW2540型聚醚砜卷式超滤膜较好,其最佳工艺参数为操作温度35℃,操作压力0.5MPa,且超滤膜透液通量较高,运行稳定。乳清蛋白粉中蛋白质含量72.40%,灰分3.85%。经红外光谱检测证明乳清蛋白粉品质得到较大程度的提高。每吨乳清废弃液中可回收乳清蛋白粉5.13kg,具有较好的经济效益及减排环保效益。     

菜籽蛋白水解物及其膜分离组分的体外抗肿瘤活性研究

应用胃肠道蛋白酶水解菜籽分离蛋白,并通过膜分离技术将水解物分离成4种不同分子量组分(1 kDa、1~3 kDa、3~5 kDa和5~10 kDa);研究了水解物和不同组分对HepG2、HeLa和MCF-7三种肿瘤细胞的体外增殖抑制作用及其氨基酸组成。结果表明:水解物及各组分对HepG2体外增殖具有较明显的抑制作用,而对HeLa和MCF-7体外增殖的抑制效果不明显;1kDa的组分对HepG2的抑制率最强(36.4%),其氨基酸组成中以谷氨酸、亮氨酸、天冬氨酸含量最高。研究认为,菜籽蛋白胃肠道蛋白酶水解物及其1 kDa的膜分离组分可能作为功能性成分用于抗肿瘤相关的功能食品和保健品的开发。