In-vitro rates of rumen proteolysis of ribulose-1,5-bisphosphate carboxylase (rubisco) from lucerne leaves,and of ovalbumin,vicilin and sunflower albumin 8 storage proteins

Laboratory systems were developed, based upon in-vitro incubations with rumen fluid, to examine the rate of proteolysis (ie degradation) of sunflower albumin 8 (SFA 8; 24% sulphur amino acids; SAA) from sunflower seeds, ovalbumin (6% SAA) from chicken egg white, ribulose-1,5-bisphosphate carboxylase (Rubisco; 3% SAA) from lucerne leaves, and vicilin (0% SAA) from pea seeds. After fractionation by SDS-PAGE, proteins were analysed by either Western blotting, using specific antibodies (SFA 8, vicilin and ovalbumin) or by Coo-massie blue staining (Rubisco). Proteolysis of the large subunit of Rubisco occurred very quickly and as two components, with half-lives of 11.6 and 1.5 h. The small subunit of Rubisco was more resistant to degradation, with a half-life of 17.3 h. Vicilin was degraded extremely rapidly (half-life 0.16 h). SFA 8 and ovalbumin both showed resistance to degradation, but by two different mechanisms. Ovalbumin was not degraded at all during the initial 16 h of incubation, but then degraded with a half-life of 8.7 h. SFA 8 (mol wt 12 100) disappeared very rapidly, with a half-life of 3.0 h. The degradation of SFA 8 was associated with the appearance of a polypeptide (mol wt 8000), which was extremely resistant to degradation, with a half-life of 69.3 h. It was concluded that both the number of disulphide linkages and tertiary structure were important in determining resistance of proteins to rumen degradation, and that incorporation of SFA 8 and ovalbumin proteins into forages using genetic engineering techniques would be likely to increase the quantity of SAA by passing rumen fermentation.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Effect of condensed tannins prepared from several forages on the in vitro precipitation of ribulose-1,5-bisphosphate carboxylase (Rubisco) protein and its digestion by trypsin (EC 2.4.21.4) and chymotrypsin (EC 2.4.21.1)

A series of in vitro experiments was undertaken to determine the extent to which Sephadex LH-20 treated extracts from a range of temperate forages precipitated ribulose-1,5-bisphosphate carboxylase (Rubisco) and affected the enzymatic hydrolysis of Rubisco protein by trypsin and chymotrypsin at a range of pH values. Rubisco was chosen because it represents the principal dietary protein for ruminants fed fresh forages. Condensed tannins (CT) or proanthocyanidins (PA) are routinely purified by chromatography using Sephadex LH-20 as a matrix. However, these extracts contained non-CT phenolics together with PA so the term ‘CT extract’ was preferred to ‘PA’ to describe the extracts. The in vitro precipitation of Rubisco provided a means to compare the reactivity of the CT extracts. The amount of CT extract required to precipitate all the Rubisco in 10 μg of total soluble leaf protein from white clover (Trifolium repens) when this protein was incubated with CT extracts of Lotus corniculatus, L pedunculatus and sainfoin (Onobrychis viciifolia) was similar, with between 25 and 50 μg of extract required. The CT extract of sulla (Hedysarum coronarium) also precipitated all the Rubisco, however this only occurred with 50 μg of the extract. The CT extract of dock (Rumex obtusifolius) precipitated all the Rubisco when 5 μg of extract or greater was incubated with total soluble leaf protein. However, the differences between the reactivity of all these CT extracts at a range of pH values appeared to be small. Condensed tannin extracts of L corniculatus and L pedunculatus partially inhibited the hydrolysis of Rubisco by trypsin and chymotrypsin to a similar extent, but the extent of the inhibition was affected by pH. The inhibition was greater at pH 6·0 than 7·0, whilst at pH 8·0, CT extracts had little or no affect on trypsin and chymotrypsin. It was concluded that, although the precipitation of Rubisco provided an ideal method for comparing CT extracts, reactivity alone was unlikely to account for the differences in nutritive value that occur with forages containing CT. © 1998 SCI.     

Analysis of iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage from a Japanese pyrite mine by use of ribulose-1, 5-bisphosphate carboxylase/oxygenase large-subunit gene

Iron- and sulfur-oxidizing bacteria in a treatment plant of acid rock drainage (ARD) from a pyrite mine in Yanahara, Okayama prefecture, Japan, were analyzed using the gene (cbbL) encoding the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO). Analyses of partial sequences of cbbL genes from Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus strains revealed the diversity in their cbbL gene sequences. In contrast to the presence of two copies of form I cbbL genes (cbbL1 and cbbL2) in A. ferrooxidans genome, A. thiooxidans and A. caldus had a single copy of form I cbbL gene in their genomes. A phylogenetic analysis based on deduced amino acid sequences from cbbL genes detected in the ARD treatment plant and their close relatives revealed that 89% of the total clones were affiliated with A. ferrooxidans. Clones loosely affiliated with the cbbL from A. thiooxidans NB1-3 or Thiobacillus denitrificans was also detected in the treatment plant. cbbL gene sequences of iron- or sulfur-oxidizing bacteria isolated from the ARD and the ARD treatment plant were not detected in the cbbL libraries from the treatment plant, suggesting the low frequencies of isolates in the samples.     

Isolation of lactic whey proteins in the form of complexes with apple pectin

The possibility of isolating lactic whey proteins (LWP) in the form of insoluble complexes (IC) with apple pectin was studied. The effect of pH, ionic strength (mu, NaCl), temperature (T degree C), pectin weight fraction (X3%) and the total concentration of macromolecular components in the system (Ws) on isolation has been considered. The process has been characterized by LWP yield in the composition IC--P, percentage and the extent of protein concentration in the concentrated phase (IC)--F. The mixing of lactic whey with a pectin solution usually yielded an IC (10% less than or equal to X3 less than or equal to 90%). The dependence of P on X3 is of an extreme nature with a maximum of 85% at X3 = 60%. The following conditions correspond to the maximum LWP yield (90%) in the complex composition: pH 3.4, mu = 0.01, T = 5-20 degrees C, X3 = 60%, Ws = 0.5%. At compositions of the system corresponding to the maximum P value (X3 = 60%) practically all the LWP fractions are present in the concentrated phase. If X3 much greater than 60% or X3 much less than 60% alpha-lactalbumin is practically absent in the concentrated phase. Usually, minimum F values (2.5-4.0) correspond to maximum protein yield at X3 = 60%. At X3 greater than 70% and X3 less than 50% F values may be considerably higher (20 times and more). A decrease in the pectin methylation degree from 56.7% down to 15.4% does not affect F. The maximum protein yield (94%) occurs when low methylated pectin is used. The character of the dependence of F on X3 is explained according to similar processes of complex gel formation and the processes of gel formation in polymer solutions.     

Interaction of lysine with pectin

The interaction of l-lysine with citrus pectin was studied in a model system to determine under what conditions it would occur and whether it would be of physiological significance. Thirty-nine per cent of the lysine was non-diffusible at pH 7, when the pectin concentration was 0·5% and the lysine concentration was $\text{2 × 10}{}^{\mathrm{?3}}\text{M}$. Of this, 29% was restricted due to the Donnan effect and the remainder was specifically bound. Increasing the pH (in the range 6–8) and the pectin concentration both increased the non-diffusible lysine. Increasing the ionic strength and the degree of esterification both decreased the non-diffusible lysine. It was concluded that lysine would not bind to pectin in the intestine because of the high salt concentration there.     

Conformational stability of ribulose 1,5 biphosphate carboxylase from tobacco leaves according to the differential scanning microcalorimetry

Protein conformation is one of the key factors which determine the behaviour of a protein in solution. During the isolation and processing into foodstuffs proteins may undergo significant conformational changes, involving structural changes on different levels — from the secondary level to the submolecular one. One of the most urgent problems related to food protein processing is the regulation of the conformational changes of proteins in order to obtain steady structures with desirable functional properties [1]. The present work is concerned with the effect of pH on the conformational stability of Ribulose 1,5-Bisphosphate Carboxylase (RBPC) from tobacco leaves. This protein constitutes 50% of the soluble proteins in green leaves of plants [2]. RBPC is an oligomeric enzyme which consists of 16 noncovalently linked subunits: large and small with a molecular mass of 55 and 14 kDa, respectively.     

Effect of pH on the properties of soy protein–pectin complexes

The interactions of a commercial soy protein isolate (SPI) and a 2:1 SPI:high methoxy pectin (PEC) complex were evaluated over a range of pH values (3-7). The SPI formed very large (> 50 ??m) and largely insoluble aggregates (< 10%) close to its isoelectric point (IEP, pH 4 and 5) and smaller, more soluble (> 80%) particles at higher and lower pH values. The addition of PEC increased the solubility of SPI close to its IEP (pH 4 and 5) and prevented the formation of very large aggregates. However, PEC reduced the solubility of SPI at higher and lower pH values presumably via a depletion mechanism. The ??-potential of diluted SPI dispersions decreased from positive to negative with increasing pH, passing through zero at pH 4.6, the isoelectric point (IEP) of the protein. At pH < 6, the addition of PEC reduced the charge of the protein suggesting the formation of a complex while at pH 6 or 7 there was no evidence of complex formation. The increased SPI solubility in the IEP in the presence of PEC is probably due to the formation of charged complex which do not aggregate while the decreased solubility of protein in the presence at high and low PEC is probably due to the formation of insoluble complexes and a depletion interaction respectively. Thermal treatment (30 min, 90 °C) enhanced the solubility of the SPI:PEC complexes close to the IEP (pH 4 and 5), but reduces it at low pH (pH 3). The SPI:PEC complexes could be manufactured in the form of a beverage at pilot scale where their solubility was enhanced by homogenization.     

Formation of biopolymer particles by thermal treatment of β-lactoglobulin–pectin complexes

The purpose of this study was to prepare and characterize biopolymer particles based on thermal treatment of protein–polysaccharide electrostatic complexes formed from a globular protein (β-lactoglobulin) and an anionic polysaccharide (beet pectin). Initially, the optimum pH and pectin concentration for forming protein–polysaccharide complexes were established by mixing 0.5 wt% β-lactoglobulin solutions with beet pectin (0–0.5 wt%) at different pH values (3–7). Biopolymer complexes in the sub-micron size range (d = 100–300 nm) were formed at pH 5.0 and 0.1 wt% pectin. These particles were then subjected to a thermal treatment (30–90 °C at 0.8 °C min⁻¹). The presence of pectin increased the thermal aggregation temperature of the protein, although aggregate formation was still observed when the protein–polysaccharide systems were heated above about 70 °C. The impact of pH (3–7) on the properties of heat-treated biopolymer particles (83 °C, 15 min, pH 5) was then established. The biopolymer particles were stable to aggregation over a range of pH values, which increased as the amount of pectin was increased. The biopolymer particles prepared in this study may be useful for encapsulation and delivery of bioactive food components, or as substitutes for lipid droplets.     

Reaction of apple pectin with ammonia

Amidated apple pectins were prepared in aqueous and aqueous-alcoholic media, and the factors influencing both ammonolysis and hydrolysis of the ester groups of apple pectin by ammonia were studied.
In aqueous solution, the extent of ammonolysis and hydrolysis was very dependent upon ammonia concentration. In aqueous alcohol, the products were also dependent upon the particular alcohol and its concentration, increasing concentrations suppressing hydrolysis; the overall rate of reaction is much slower than in the absence of alcohol. The product balance between amonolysis and hydrolysis of the ester groups is influenced by the polarity of the medium, and by the concentration of NH⁺₄ and OH⁻.
It was shown that the extents of ammonolysis and hydrolysis of the ester groups are significantly affected by the polarity of the medium and the acid-base equilibrium of the interaction of ammonia with water, i.e. NH₃+H₂O ↔ NH⁺₄+OH⁻. The content of free ester and amidated carboxyl groups in the pectin can be regulated by varying the concentration of ammonia, temperature, reaction time and the polarity of the medium. The rate of enzymic hydrolysis of amidated pectins decreases with increasing amide content. A mechanism for the reaction is proposed.     

Emulsifying properties of protein–pectin complexes and their use in oil-containing foodstuffs

The emulsifying properties of proteins have been well studied as they are important for the preparation of creams, mayonnaises and other oil/fat-containing foodstuffs. The emulsifying action of a protein is not always sufficient to obtain stable emulsions of good quality. The use of chemical stabilisers in the food industry is not desirable. One of the best ways to improve the quality of emulsions and to produce emulsions with high nutritive value is to use protein-polysaccharide complexes as emulsifiers. Varying the protein-polysaccharide ratio in the complex, and also the kind of the polymers, would vary the quality and the nutritional value of the foodstuff. Four non-conventional protein preparations were tested as emulsifiers and their emulsifying properties were improved by the addition of pectin. This makes it possible to create new foodstuffs with low oil content and high nutritional value.     

Stabilisation of acidified skimmed milk with HM pectin

HM pectin was used to stabilise acidified milk drinks (AMD) against flocculation of milk protein. Two temperatures (16 and 27 °C) were used for acidification in order to obtain AMD of different average particle size. Stable samples were diluted with either serum simulating buffer (SSB) or water, and for these samples the adsorption of the pectin onto the casein aggregates was measured using HPLC. The particle diameter, viscosity and sedimentation, as well as the viscoelastic properties, were used to evaluate the amount of pectin needed to stabilise AMD. Additionally, confocal laser scanning microscopy was used with fluorescence recovery after photobleaching (FRAP) to assess protein mobility in samples. Upon diluting with water an increased adsorption of pectin onto the casein aggregates was observed and the stability decreased. In both water and SSB, a certain amount of pectin was firmly-possibly irreversibly-bound to the casein aggregates, and it could be inferred that the adsorbed pectin formed a multilayer on the casein aggregates. Even though the presence of a weak gel network was expected, no gel could be demonstrated in stable AMD by FRAP or the applied rheological measurements. We thus considerer it unlikely that formation of a network is solely responsible for the stability in AMD and propose a stabilising mechanism resembling steric stabilisation combined with secondary adsorption.     

高甲氧基果胶在稳定调配型酸乳体系中的研究

研究了高酯果胶在稳定调酸乳体系中的作用,并从粒径,黏度,沉淀率和热稳定性几个方面来评价稳定调酸乳饮料体系的果胶用量。研究发现不同的酸化温度对果胶和酪蛋白相互作用有影响,这对长货架期和短货架期酸乳饮料的研发都有实际指导作用。     

螯合剂协同微波法提取百香果皮果胶及果胶膜的制备

以六偏磷酸钠为鳌合剂,采用微波法提取百香果皮果胶,通过单因素试验和正交法优选,确定螯合剂辅助微波法提取百香果皮果胶的最优工艺条件为:加入0.3%含量的六偏磷酸钠,料液比为1∶20(g/m L),p H为3,微波功率为700 W,微波时间为4 min,果胶产率达13.72%。以百香果皮果胶与藕粉、甘油共混制备果胶可食膜,考察果胶/藕粉比例,甘油对膜的阻隔性能,机械性能及热封强度等的影响,结果表明,膜的配方为果胶/藕粉1/1,甘油添加量0.1%,制得的果胶膜的阻隔性能和机械性能较好。     

纤维素酶辅助提取橘皮中果胶类化合物

本文以橘皮为原料,研究用纤维素酶辅助提取橘皮中果胶的工艺,分别讨论了温度、pH值、时间、料液比、纤维素酶用量等因素对果胶提取率的影响,通过单因素及L16(45)正交实验,获得了橘皮中提取果胶的最佳条件,即温度55℃,pH7.0,提取时间2.5h,料液比1∶20,纤维素酶用量0.25%,在此条件下果胶提取率可达17.31%。     

Formation of electrostatic complexes using sodium caseinate with high‐methoxyl pectin and carboxymethyl cellulose and their application in stabilisation of curcumin

Despite many reported bioactivities of curcumin, its application is limited due to its low bioavailability, solubility and stability. Proteins have been reported to stabilise curcumin in aqueous media, and stabilisation of curcumin could be enhanced when proteins form an electrostatic complex with polysaccharides. In this study, electrostatic complexes of sodium caseinate (NaCas) were prepared using high‐methoxyl pectin (HMP and NaCas‐HMP) and carboxymethyl cellulose (CMC and NaCas‐CMC). NaCas to polysaccharide ratio of 1:2 resulted in the lowest turbidity and sedimentation. The electrostatic complexes were more stable than native NaCas against pH change and ionic strength. Binding of curcumin to NaCas and the electrostatic complexes were confirmed by UV‐vis and fluorescence spectra and Fourier transform infrared spectroscopy (FT‐IR). The electrostatic complexes showed a higher binding constant and protected curcumin better than the native NaCas. This study suggests that the electrostatic complexes may be a superior carrier to NaCas at an acidic environment.     

盐析法提取柚子皮果胶工艺的研究

研究了用盐析法从柚皮中提取果胶的工艺条件。结果表明,盐析法提取柚皮果胶的最佳工艺条件为提取液pH值1.5,提取温度90℃,提取时间50min,料液比1∶25（g∶mL）,饱和明矾溶液与水解液体积比1∶1,盐析温度60℃,盐析时间50min,盐析pH值4.0。在此工艺条件下,果胶得率为18%。     

Formation and characterization of lactoferrin/pectin electrostatic complexes: Impact of composition,pH and thermal treatment

Protein–polysaccharide complexes may be used for the development of delivery systems with applications in several industries. In the present work, the interaction of lactoferrin (LF, 0.2wt%) with a High-Methoxyl pectin (0.005–0.15wt%) in aqueous solutions was studied at different pH values (2–7) and temperatures (30–90 °C) at low ionic strength. ζ-potential and light-scattering techniques were used to provide information about the electrical charge and aggregation of individual biopolymers and complexes. At pH 7, the electrical charge went from positive to negative when increasing amounts of pectin were added to the LF solution, which was attributed to the formation of an electrostatic complex. These complexes remained soluble (low turbidity) from pH 7 to 3.5, but became turbid between pH 3.5 and 2, due to charge neutralization and bridging effects. At pH 7, the stability of LF–pectin complexes to aggregation during heating was much better than LF alone. The results of this study should provide information that will facilitate the utilization of lactoferrin as a bioactive component in food systems.     

苜蓿营养挂面的研制

利用食物中营养素间的互补作用原理,在挂面生产中添加适量的苜蓿蔬菜糊,制成营养丰富的苜蓿挂面--绿色翡翠挂面;从营养成分和色泽方面对苜蓿添加量的选择作进一步的研究;以苜蓿为辅料制作的营养保健挂面具有色泽鲜亮、柔和,风味特殊,营养丰富,保健功效好等特点,是理想的天然营养保健食品,具有较高的营养与推广价值。