乳清蛋白抗菌肽的分离纯化及活性

以乳清蛋白为原料,酶解制备具有抗菌能力的生物活性肽。采用超滤、葡聚糖凝胶层析色谱、反相高效液相色谱法对酶解液进行分离纯化,并对分离产物的氨基酸组成进行分析。结果表明:葡聚糖凝胶色谱纯化最佳流速2 mL/min,最佳样品质量浓度15 mg/mL;再由反相高效液相色谱分离的高活性抗菌肽,抑菌圈直径达到31.33 mm。氨基酸分析结果显示,高活性抗菌肽碱性氨基酸和疏水性氨基酸含量最多,分别为42.69%和37.39%。     

HPLC测定饲料源小肽产品中赖氨酸含量

将蛋白饲料源小肽产品按照GB/T 18246-2000中酸水解法进行水解及相应的处理,获得氨基酸混合液与2,4-二硝基氯苯(CDNB)衍生化反应后,其中赖氨酸生成二硝基氯苯赖氨酸(DNP-LYS),经日本岛津SPD-10AVG高效液相色谱(HPLC),C18柱分离,UV350 nm检测,衍生后氨基酸混合物得到较好的分离,赖氨酸最低检出量2μg.     

Porcine intestinal yeast species,Kazachstania slooffiae,a new potential protein source with favourable amino acid composition for animals

There is little information about Kazachstania slooffiae which dominates among other yeasts in the pigs’ intestine. Therefore, the aims of this study were to characterise the yeast cell contents and to investigate which nitrogen sources, organic acids and alcohols may be utilised or produced by this species. The results showed that, K. slooffiae could use urea, ammonium sulphate, peptides and single amino acids and produce thereby ethanol and formic acid. However, this yeast did not metabolise amino acids, lactic, butyric, propionic and acetic acids as sole carbon source. Using a global metabolite profiling approach employing gas chromatography and high‐resolution liquid chromatography mass spectrometry, was found that the amount of peptides and dehydroascorbic acid considerably increased in the fermentation residues after yeast cultivation. It is noteworthy that the cells of K. slooffiae had higher contents of nitrogen and total amino acids (especially lysine) than the cells of nutritional yeast (Saccharomyces cerevisiae). This study indicates that due to potential production of peptides and formic acid in the intestinal tract, K. slooffiae might have an impact on the gut health. Moreover, from a nutritional standpoint, the cells of this yeast can be a good source of protein with useful amino acid composition for animal.     

Dietary animal proteins alter monoamine metabolism in the brain

Several amino acids have effects on mental function, including sedative, antidepressant‐like and anxiolytic‐like effects. However, the influence of integrated amino acid nutrition as protein constituents on mental function remains unclear. Therefore, the purpose of the present study was to compare the influence of chicken, pork and beef protein extracts on brain monoamine metabolism in mice. Changes in monoamine levels and their turnover rates in the brain were induced by different protein sources. In particular, chicken protein group showed the highest norepinephrine levels in the hippocampus and hypothalamus, and beef protein extract caused an activation of the serotonergic system in the hypothalamus, although there were no significant differences in amino acid compositions of these protein extracts. Therefore, it was revealed that amino acid compositions in dietary protein did not induce alteration in monoamine metabolism. However, there were differences in small molecular peptides, such as creatine, carnosine and anserine levels in animal protein extracts. In conclusion, monoamine metabolism was altered by dietary protein sources. However, it was indicated that the alteration in monoamine metabolism may be independent from amino acid compositions in dietary protein. In addition, alteration in monoamine metabolism depending on the dietary protein sources may be induced by small molecular peptides.     

Isolation and characterization of C-reactive protein and serum amyloid P component from bovine serum

C-reactive protein (CRP) and serum amyloid P component (SAP), which are known as acute phase reactants in human and many other animals, were purified from cow sera. Affinity chromatography using HE agarose gel was the most effective method to isolate both CRP and SAP from a large volume of bovine serum. Separation of CRP and SAP from the mixed preparation could be performed by DEAE-cellulose column chromatography, gel permeation HPLC using TSK-G3000SW or affinity chromatography using phosphorylcholine derivatives of bovine serum albumin-conjugated Toyopearl HW 65. Bovine CRP and SAP were identified as genuine CRP- and SAP-class proteins by their cross reactivities with anti-human CRP and anti-human SAP, respectively, and by their homology in amino acid compositions compared with those of human CRP and SAP, respectively. Bovine CRP moved slower than beta-globulin, and bovine SAP moved in the beta-globulin region in agarose gel electrophoresis. Both of them gave single bands in native polyacrylamide gel electrophoresis (PAGE). Bovine CRP and SAP molecular weights were estimated to be 100,600 and 109,500 daltons respectively, by sedimentation equilibrium analysis. Bovine CRP showed 23K dalton subunits by sodium laurylsulfate-PAGE and bovine SAP showed 28K and 32K dalton subunits, both of which were glycosylated and had identical amino acid compositions, indicating that both CRP and SAP molecules are pentamers. In fact, they appeared to have pentameric disk-like configurations in electronmicroscopical examination.     

Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase

This study aims to identify peptides with angiotensin-I converting enzyme (ACE) inhibitory activity in hydrolysate from chicken leg bone protein hydrolyzed with alcalase for 4 h (A4H). The hydrolysate has demonstrated potent in vitro ACE inhibitory activity, and has been shown to attenuate the development of hypertension and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). A4H is competitive for ACE and was separated using high-performance liquid chromatography (HPLC) with a gel filtration column (Superdex Peptide HR 10/30). The results show that A4H is a mixed non-competitive inhibitor. Eighteen fractions were detected after separation of A4H, and most of them showed ACE inhibitory activity. Five fractions with strong ACE inhibitory activities (above 50%) were labeled from A to E. In addition, there were 10 peptides, consisting of 5–10 amino acid residues that were identified from fraction D that exhibited the strongest ACE inhibitory activity. Three of the identified peptides corresponded to peptides derived from collagen type I and chicken muscular protein. It is revealed that A4H has several peptides that possess ACE inhibitory activities.     

Correlation between skeletal muscle fiber type and free amino acid levels in Japanese Black steers

Free amino acids are important components of tastants and flavor precursors in meat. To clarify the correlation between muscle fiber type and free amino acids, we determined the concentrations of various free amino acids and dipeptides in samples of different muscle tissues (n = 21), collected from 26‐month‐old Japanese Black steers (n = 3) at 2 days postmortem. The proportions of the myosin heavy chain (MyHC), slow (MyHC1) and fast (MyHC2) isoforms were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The contents of free amino acids and dipeptides were measured by high performance liquid chromatography (HPLC). The MyHC isoform composition varied among the tissue samples. The MyHC1 proportion ranged from 6.9% ± 3.9% to 83.3% ± 16.7%. We confirmed that there was a strong positive correlation between MyHC1 composition and total free amino acid concentrations, including those for two dipeptides. Among the 31 measured free amino acids and dipeptides, 11 showed significant positive correlations and five showed significant negative correlations with MyHC1 composition. These results suggest that a high MyHC1 content induces high free amino acid contents in bovine muscles possibly because of greater oxidative metabolism. This high level of free amino acids could contribute to the intense flavor of meat that is rich in slow‐twitch fibers.     

Characterization of peptides in ensiled alfalfa treated with different chemical additives

Effects of different chemical additives on peptide composition in ensiled alfalfa (Medicago sativa L.) were investigated by using gel filtration and determination of N characteristics. The alfalfa silages were prepared untreated (control) or with formic acid, formaldehyde or tannic acid as additives at ensiling. All additives reduced non‐protein N (NPN), ammonia nitrogen (NH₃‐N) and amino acid N (AA‐N) in the ensiled forage, and the most effective reduction of NPN and AA‐N was observed in the formaldehyde‐treated silages. Peptides in the control silage were mainly dipeptides to peptides with five amino acid residues. Most peptides in the formic acid‐treated silage contained 4–12 amino acid residues. Although most peptides in the formaldehyde‐treated silages contained 4–6 amino acid residues, there was a considerable proportion of peptides with 7–11 amino acid residues. Tannic acid had little effect on peptide size of ensiled alfalfa extract in which most peptides contained 5–6 amino acid residues. Peptide size in formic acid‐treated alfalfa silage was greater than that in the other treatments. Addition of formic acid and formaldehyde not only increased the peptide concentration in alfalfa silage, but enlarged the peptide size.     

Palatability and physicochemical properties of sausages prepared via lactic acid fermentation and drying at low temperature

The present study aimed to assess the palatability of sausages undergoing low‐temperature fermentation and drying process. Lactobacillus sakei D‐1001 or Lactobacillus salivarius A001 were used as starter cultures for fermentation, and the following properties of the sausages were investigated: colony‐forming units of lactic acid bacteria; concentrations of lactic acid, protein, peptides, and free amino acids; distribution of protein; composition of free amino acids; and physical properties and taste. Alterations in the composition of proteins, peptides, and free amino acids as well as in various physical properties were caused by fermentation by lactic acid bacteria. A sensory test indicated that the palatability of the fermented sausages was greater than that of the non‐fermented sausages, particularly in terms of hardness and juiciness. This was considered to be due to protein degeneration and changes in the physical properties of the sausages as a result of fermentation by lactic acid bacteria. However, the taste of the fermented sausages was sourer than that of the non‐fermented sausages, and therefore, inferior. Our study revealed that the palatability of the sausages in terms of hardness and juiciness were increased by low‐temperature fermentation by lactic acid bacteria and the drying process.     

An endurance‐enhancing effect of peanut meal protein hydrolysate in mice: possible involvement of a specific peanut peptide

To improve the functional properties of peanut meal protein for wide utilization, hydrolysis was conducted by alcalase. Compared with saline and peanut meal protein, intragastric administration of low molecular weight (<1 kD) peanut meal peptide (PPH I) could significantly prolong swimming time, increase levels of blood sugar, non‐esterified fatty acids (NEFA) and liver glycogen and decrease blood lactate content in mice. Levels of Pro, Leu, Val and His in low molecular weight peanut meal peptides were higher significantly than those in other peanut meal protein hydrolysates. Hydrophobic amino acids, such as Pro, Tyr and His, could perhaps capture free radical and increase antioxidant capacity of peanut peptide and retard fatigue induced by free radical. After separation by HPLC, a primary peptide P1, Pro‐Glu‐Ile‐Glu‐Val, was sequenced. Its N‐terminal was Val, and it was rich in antioxidant amino acid, Pro and Ile. Levels of plasma glucose, NEFA and liver glycogen in PPH I group were higher than those in mice intragastric administration with peptide P1, and the swimming time is longer in PPH I group than in P1 group. So, the high content of P1 was one of the reason why PPH I had high endurance‐enhancing capacity.     

Studies on the incorporation of 14 C-labelled amino acids into egg proteins

The incorporation of ¹⁴C‐labelled amino acids into egg white and yolk proteins has been studied. When the labelled amino acids were given intravenously as a hydrolysate of [U‐¹⁴C]‐protein from Chlorella, 10 per cent and 7 per cent of the ¹⁴G were recovered in the whites and yolks respectively of the first nine eggs laid. Differences in the specific activities of the conalbumin, the ovalbumins, ovomucoid, “postalbumins “ and lysozyme, isolated from the first active egg, were related to differences in amino acid composition. The specific activity of each amino acid prepared from the proteins was similar between different proteins, although within each protein specific activities of different amino acids varied widely. The proportionate rate of decrease of specific activity in the plasma of amino acids essential for egg production (except glycine) was constant or almost so, but it rapidly decreased for the non‐essential ones (except serine and proline). The specific activities of the amino acids, excepting aspartic and glutamic acids, in each protein were proportional to their mean specific activities in the plasma throughout the 24‐h period in which the egg white proteins were synthesised. It is concluded that these different proteins are synthesised from a shared amino acid pool, derived from plasma, at a rate which probably remains constant throughout the egg‐laying cycle and which is proportional to their content in the albumen.     

Susceptibility difference between methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP‐28)

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin‐resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP‐28. This study aims to clarify how a few amino acid substitutions of BMAP‐28 are related to its antimicrobial activity using four analog peptides of BMAP‐28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP‐28 do not change antimicrobial activity. It was also revealed that the percentage of cis‐11‐eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP‐28.     

绢纺精练液中丝胶蛋白的氨基酸组成、相对分子量及溶解性的测定

利用氨基酸自动分析仪对从桑蚕丝条吐精炼液中回收的丝胶蛋白的氨基酸组成进行分析,采用聚丙烯凝胶电泳法对其相对分子质量进行测定,利用紫外分光光度计对其溶解性进行探究。结果表明:丝胶蛋白含有18种氨基酸,其中极性氨基酸占80.39%,丝氨酸占27.52%,天门冬氨酸占15.91%,苏氨酸7.41%;丝胶蛋白的相对分子质量集中分布于17~75 KD;丝胶蛋白溶解性优于大豆分离蛋白。     

Ileal amino-acid digestibility of wheat, autoclaved wheat and spaghetti by-products for broiler chicks

An experiment was conducted to determine true and apparent ileal amino‐acid digestibility of a native cultivar of wheat (Mahdavi), autoclaved wheat (120°C for 30 min) and spaghetti by‐products available in Iran. One hundred 21‐day‐old broiler chickens were fed a standard corn–soybean meal starter diet from day 0 to 28 post hatch. At 28 days 80 chicks were distributed according to possessing very nearly the same average bodyweight to 20 wire cages. The experimental units were allocated at random to five dietary treatments with four replicates per treatment. The basal diet contained corn and soybean meal as the major ingredients. Three test diets were formulated containing wheat, autoclaved wheat and spaghetti by‐products as the sole source of dietary protein and each test diet was combined with a basal diet 50:50 on a weight basis to form three assay diets. The apparent and true digestibility of amino acid in the test ingredients were estimated from those in the assay diets basal/test diet mixtures, using the difference method. The apparent and true amino‐acid digestibility of the test ingredients were significantly different (P < 0.01). Autoclaving of wheat increase its amino‐acid digestibility (P < 0.01). Among the test ingredients, the average ileal amino‐acid digestibility of spaghetti by‐products was higher than wheat and autoclaved wheat but it was lower than values for the basal diet (P < 0.01).     

Isolation, characterization, and quantitative analysis of C-reactive protein from horses

C-reactive protein (CRP) was isolated from equine serum by use of calcium-dependent affinity chromatography conjugated pneumococcal C-polysaccharide, anion exchange chromatography, and gel filtration. It was identified as genuine CRP by its immunochemical cross-reactivity with anti-human CRP, its homology with human CRP in amino acid composition, and its pentameric structure as revealed by electron microscopy. Purified equine CRP had a molecular weight of approximately 118,000 and was composed of 5 identical, nonglycosylated and noncovalently associated subunits with molecular weight of approximately 23,000 each. Equine CRP migrated in the region between beta- and gamma-globulin by results of immunoelectrophoresis, and its isoelectric point was about 7.0. In horses, increased CRP concentration was associated with clinical pneumonitis, enteritis, and arthritis, compared with values obtained in clinically normal horses by use of single radial immunodiffusion method. After IM administration of turpentine oil or castration, serum CRP concentration increased to 6 times higher than baseline values. Results indicate that CRP may be an acute-phase reactant protein in horses.     

Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.     

乳及乳制品中皮革水解蛋白检测方法的研究进展

本文介绍了乳及乳制品中皮革水解蛋白的几种检测方法,主要包括比色法、氨基酸自动分析仪测定法、高效液相色谱法、高效液相色谱-质谱联用法、毛细管电泳法,并对各种方法的原理及其研究情况进行了详细阐述。     

In vitro metabolism of tyrosine by rumen bacteria, protozoa and their mixture

An anaerobic in vitro study of tyrosine (Tyr) metabolism by rumen bacteria (B), protozoa (P) and their mixture (BP) was conducted using an HPLC analytical method to obtain some basic and systematic information on aromatic amino acid metabolism in the rumen. Most of the Tyr (> 96%) disappeared after 12 h incubation in B and BP, but only 58% in P. Tyr was converted mainly to p‐hydroxyphenylacetic acid (HPA) in all microbial suspensions. About 45% of disappeared Tyr in B and P, and about 35% in BP were converted to HPA. An appreciable amount of phenylalanine (Phe), 13 and 3% of disappeared Tyr, and a small amount of tryptophan (Trp), 8 and 1% of disappeared Tyr, were also produced from Tyr by rumen bacteria and protozoa, respectively. Small amounts of p‐hydroxyphenylpyruvic acid (about 4 and 6% of disappeared Tyr) were produced from Tyr in B and P, respectively. A moderately large amount of phenylacetic acid (14% of disappeared Tyr) was produced from Tyr in P which was 1.9 times higher than that in B. Phenylpropionic acid and p‐hydroxybenzoic acid were produced only in B and BP. It was concluded that the Tyr degradation ability of rumen bacteria was about 1.5 times higher than that of rumen protozoa. Degraded Tyr mainly produces HPA and then two other aromatic amino acids, Phe and Trp, which are considered essential amino acids for ruminants. Therefore, it is speculated that the requirement for Phe and Trp in ruminants may be partially fulfilled if Tyr is sufficiently supplied in rations.     

蛹虫草多糖纯化工艺研究

为优化蛹虫草多糖的纯化工艺,以蛋白去除率和多糖保留率为指标,比较Sevage法、改良的Sevage法、澄清剂法三种方法对粗多糖中蛋白杂质的去除效果。本试验依次采用DEAE－52、Sephdex G －100葡聚糖凝胶柱层析进一步分离纯化蛹虫草多糖,再经Sephdex G－200柱色谱及高效液相凝胶色谱进行纯度检验。结果显示：澄清剂法蛋白去除率为93．4％,多糖保存率为90．7％;DEAE －52柱层析得到一个洗脱峰CP ,再经Sephdex G －100柱层析后得到两个洗脱峰CP－1和CP－2。对CP－1和CP－2进行纯度检验,发现两者皆为均一性多糖。