牛黑素皮质素受体1(MC1R)基因与毛色表形的研究

牛MC1R基因不仅与毛色有关, 而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛, 中国荷斯坦红白花牛, 鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(E^D, E⁺, e)。中国荷斯坦黑白花牛主要是E^D和E⁺等位基因(E^D=0.12、E⁴=0.80); 渤海黑牛也主要是E^D和E⁺等位基因(E^D=0.52、E⁺=0.47); 中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E⁺/e基因型。由此推测E^D和E⁺等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

牛黑素皮质素受体1(MCIR)基因与毛色表型的研究

牛MC1R基因不仅与毛色有关,而且与牛乳中乳蛋白的含量有关。利用PCR-RFLP和DNA测序技术分析了中国荷斯坦黑白花牛,中国荷斯坦红白花牛,鲁西黄牛和渤海黑牛共4个品种的MC1R基因。共检测出3种等位基因(ED,E ,e)。中国荷斯坦黑白花牛主要是ED和E 等位基因(ED=0.12、E =0.80);渤海黑牛也主要是ED和E 等位基因(ED=0.52、E =0.47);中国荷斯坦红白花牛和鲁西黄牛大多为e等位基因(e=0.95)。中国荷斯坦红白花牛和鲁西黄牛还存在E /e基因型。由此推测ED和E 等位基因导致黑色素合成。另外发现牛MC1R基因编码区725处存在一重要的SNP(单核苷酸多态性)。     

Characteristics of Korean-Saanen goat milk caseins and somatic cell counts in comparison with Holstein cow milk counterparts

Characteristics of α- and β-casein fractions in the milk of Korean-Saanen goats were compared with those of Holstein cow milk using capillary electrophoresis (CE) analysis. The αs₁-CN content of the Saanen goat milk samples varied from 2.4% to 9.3% of total proteins. Total αs-CN content of the goat milk varied from 10.1% to 17.0%. Total β-CN content containing β₁-CN and the β₂-CN varied from 49.6% to 61.0% of total proteins. Average αs-CN to β-CN ratio of the Saanen goat milk from different farms was 0.24 ± 0.04, ranging from 0.17 to 0.33. The αs-CN (αs₁-CN + αs₀-CN) to β-CN (β_A1-CN + β_A2-CN) ratio of Holstein cow milk was 0.81, which was much higher than that of Korean-Saanen goat milk. The goat milk samples having more than 1.5 million cells/ml somatic cell counts (SCC) contained higher αs-CNs (P < 0.01) and lower β-CNs (P < 0.05) contents than milks with <1.5 million SCC. This resulted in a higher αs-CN to β-CN ratio (P < 0.01) in the milk with >1.5 million SCC.     

Association of SCD1 and DGAT1 SNPs with the intramuscular fat traits in Chinese Simmental cattle and their distribution in eight Chinese cattle breeds

Intramuscular fat (IMF) is a key parameter for evaluation of nutritional quality of beef, with its endogenous synthesis regulated by stearoyl CoA desaturase (SCD1) and diacylglycerol-acyl transferase 1 (DGAT1) genes in cattle. The object of this research was to evaluate the effect of SCD1 and DGAT1 polymorphisms on IMF trait in beef cattle and to estimate the frequency distribution of SNPs in the two genes in Chinese cattle populations. The SCD1 and DGAT1 polymorphisms were detected by PCR-single strand conformation polymorphism (PCR-SSCP) method in Chinese Simmental cattle and their associations with IMF traits were analyzed using the general linear model (GLM). The frequency distribution of SNPs in SCD1 and DGAT1 genes were detected by PCR-SSCP method and analyzed in seven other cattle populations. The results showed significant associations of SNPs SCD1-878, SCD1-762, and DGAT1 10433 and 10434 with IMF (%) and shearing force values (SFV; kg) in Chinese Simmental cattle. A haplotype combining SCD1-878C, SCD1-762T, and DGAT1 10433 and 10434-GC had the highest IMF, marbling score and shearing force. The polymorphic investigation indicated that the frequency of SCD1-878C or SCD1-762T was significantly higher in Chinese southern cattle (Leiqiong, Yunnan High pump, BMY or Minnan Cattle) than in Chinese northern cattle (Chinese Simmental, Luxi Cattle, Bohai Black or Chinese Holstein), while the frequency of DGAT1 10433 and 10434-GC in Chinese indigenous breed (Leiqiong, Yunnan High pump, BMY, Luxi Cattle, Bohai Black, or Minnan Cattle) was significantly lower than breeds with imported blood (Chinese Simmental or Chinese Holstein). These findings demonstrated that both the SCD1 and DGAT1 SNPs were prospect genetic markers for IMF traits, and the SCD1 SNPs could be used as a genetic marker for southern or northern blood in Chinese cattle.     

Associations between newly discovered polymorphisms in the Bos taurusgrowth hormone receptor gene and performance traits in Holstein–Friesian dairy cattle

Variations in the growth hormone receptor (GHR) gene sequence are associated with performance traits in cattle. For example, the single nucleotide polymorphism (SNP) F279Y in transmembrane exon 8 has a strong association with milk yield. In this study, 32 previously unreported, putative novel SNPs (31 in the 5′ non‐coding region) were identified by resequencing ～19 kb of the GHR gene in genomic DNA from 22 cattle of multiple breeds. A population of 848 Holstein–Friesian AI sires was subsequently genotyped for the 32 putative novel SNPs and seven published SNPs (including F279Y, one in exon 1A promoter and five in exon 10). Associations between each segregating SNP and genetic merit for performance were quantified in the 848 Holstein–Friesians using weighted animal linear mixed models. Six of the published SNPs and seven of the novel SNPs were associated with at least one of the traits – milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score, calving interval, survival and growth and size traits. Even when the allelic substitution effect (P < 0.001) of F279Y was accounted for, the allelic substitution effect of one of the novel SNPs (GHR4.2) in the 5′ non‐coding region of GHR was associated with a lactation milk yield of 37.46 kg (P < 0.001). GHR4.2 and F279Y were not in linkage disequilibrium (r² = 0.00, D’ = 0.04) in the 848 Holstein–Friesians, indicating that their association with milk yield was independent.     

Control of estrus in bos indicus and bos taurus heifers with prostaglandin F2 alpha

Lutalyse^*, a commercial preparation of prostaglandin F_2a was injected intramuscularly into nulliparous heifers of the German Brown, Holstein—Friesian and White Fulani cattle. Two injections, 25mg/animal each time, were given at intervals of 12 days. All the heifers were inseminated 80h after the second injection. Heifers that were cycling responded well to the treatment and had a higher conception rate than the controls.     

The relative influence of temperature and humidity on cutaneous function in Bos indicus and Bos taurus females

The relative importance of dry- and wet-bulb temperatures on cutaneous function inBos indicus andBos taurus females under humid tropical climatic conditions was evaluated. The parameters investigated were sweating rate and skin temperature, while the species utilised were zebu White Fulani (Bos indicus) and German Brown and German Black and White (Bos taurus).The sweating rate, irrespective of breed, differed with the site of sampling and was more influenced by dry-bulb (59%) than by wet-bulb temperature (41%). Skin temperature responded more to wet-bulb temperature in White Fulani and German Black and White cattle, but not in German Brown cattle.It is concluded that the response of the animals, with respect to sweating, was similar but that the efficiency of sweating, judged by the lowering of skin temperature, was higher inBos indicus than inBos taurus. This, in part, may explain the higher degree of comfort demonstrated byBos indicus under tropical conditions.     

Complex vertebral malformation in Holstein cows in Japan and its inheritance to crossbred F1 generation

Complex vertebral malformation (CVM) is an autosomal recessive inherited defect in the Holstein breed. It causes intra-uterine mortality leading to repeat breeding and involuntary culling of cows. This study was carried out to show the prevalence of CVM carriers among Holstein cows in five dairy farms in Hiroshima Prefecture, South Western Japan and whether the defect could be inherited to crossbred F1 generation, when Japanese Black semen was used to inseminate a Holstein cow. Two hundreds Holstein cows were used in the study. Blood samples were collected from all cows in a heparinized tubes and genomic DNA was extracted with a commercial kit. Apart of the bovine solute carrier family 35 member 3 (SLC35A3) gene around the suspected mutation (G to T) was amplified with an allele specific polymerase chain reaction (AS-PCR). For DNA sequencing, PCR products of 522 bp were obtained from the genomic DNA of the cows. Out of 200 examined cows, 26 cows (13.0%) were found to be CVM carriers. Wild type cows showed amplification with the G-specific primer pair but not with the T-specific primer pair, while carrier ones showed amplification of both the G- and T-specific primers, exhibited two DNA bands of 395 bp. Based on the clinical history of the cows, lowered reproductive performance was noticed in carrier ones. Out of four crossbred F1 calves (Holstein x Japanese Black), two were CVM carrier. Crossbred F1 could inherit the CVM defect when Japanese Black semen used to inseminate a carrier Holstein cow. In conclusion, the study reports the occurrence of CVM among Holstein cows in Hiroshima Prefecture, Japan; moreover, it describes the first cases of CVM in crossbred F1 generation.     

Angiotensin I-Converting-Enzyme-Inhibitory and Antibacterial Peptides from Lactobacillus helveticus PR4 Proteinase-Hydrolyzed Caseins of Milk from Six Species

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine α_S1-casein (α_S1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine α_S1-CN f1-6 and α_S2-CN f182-185 and f186-188; caprine β-CN f58-65 and α_S2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC₅₀) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC₅₀s were confirmed. An antibacterial peptide corresponding to β-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.     

Hydrolysis of Casein-Derived Peptides αS1-Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase

Peptides derived from hydrolysis of α_S1-casein(f1-9) [α_S1-CN(f1-9)] and β-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (ΔpepC, ΔpepE, ΔpepN, ΔpepO, and ΔpepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.     

Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and α_S1-casein (α_S1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards α_S1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and α_S1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides α_S1-CN (f1-9), α_S1-CN (f1-13), and α_S1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and α_S1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks (<Emphasis Type="Italic">Lassi</Emphasis>) Fermented by <Emphasis Type="Italic">Lactobacillus acidophillus</Emphasis> with Consideration of Incubation Period and Simmering Treatment

The lassi, fermented milks product containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus acidophilus NCDC-15 and the incubation period and simmering effect was also optimized for production of ACE-inhibitory peptides. The time–temperature combination for the heat treatment was optimized using RSM. The biological activity was measured in the supernatant of the fermented milk after centrifugation. The lowest IC₅₀ values for the inhibition of angiotensin-converting enzyme (ACE) was found 28.9 ± 0.95 μg protein/ml in the supernatant of milk fermented by L. acidophilus and heated at 78 °C for 10 h. The fractions which showed the highest ACE-inhibitory indexes were further purified by different techniques including solid phase extraction, RP-HPLC and FPLC and the related peptides were identified by LC–MS/MS using the Ultimate 3000 nano HPLC system (Dionex) coupled to a 4000 Q TRAP electro-spray ionization mass spectrometry. The high ACE-inhibitory activity containing fractions of the milk fermented by L. acidophilus contained the sequences of b-casein (b-CN) fragment. The fraction-III showed minimum IC₅₀ value i.e. 14.57 ± 0.72 μg/ml compared with fraction-I and fraction-II. Among these peptides 14 peptides have been identified from the fraction-I of the lassi prepared from L. acidophilus i.e. β-CN f47–56, β-CN f47–57, β-CN f199–209, β-CN f176–182, β-CN f176–183, β-CN f176–184, β-CN f1–7, β-CN f57–68, β-CN f166–175, β-CN f195–206, β-CN f195–207, β-CN f195–209, β-CN f94–106 and β-CN f169–176 showed partially or completely homology to that the milk protein bioactive peptides having ACE inhibitory. The two peptides KVLPVPQK (β-CN f169–176) and YQEPVLGPVRGPFPIIV (β-CN f193–209) have the same sequence as ACE inhibitory peptides (Maeno et al. in J Dairy Sci 79(8):1316–1321, 1996; Yamamoto et al. in J Dairy Sci 77:917–922, 1994b).     

Induction of twinning in Holstein and Japanese black cows by ipsilateral frozen embryo transfer

Induction of twinning by ipsilateral nonsurgical transfer of two frozen-thawed Japanese Black bovine embryos to each of 20 Holstein and 26 Japanese Black cows, that had been kept under a stable in private farm conditions, was examined. The cows were monitored every 20 days from Day 25 to Day 65 of gestation for pregnancy and fetus survival (estrus is Day 0). Seventy-five per cent (15 of 20), 65.0% (13 of 20) or 60.0% (12 of 20) and 61.5% (16 of 26), 53.8% (14 of 26) or 50.0% (13 of 26) of Japanese Black cows were diagnosed pregnant at 25, 45 and 65 days after transfer by ultrasonic echography. Embryonic losses were observed between Days 25 and 65 in 29.2% (7) Holstein and 31.8% (7) Japanese Black cows. The twin pregnancy rate in Holstein and Japanese Black cows decreased with time; 60.0% (9 of 15) vs. 37.5% (6 of 16) at Day 25; 53.8% (7 of 13) vs. 28.6% (4 of 14) at Day 45 and 41.7% (5 of 12) vs. 15.4% (2 of 13) at Day 65. At calving, Holstein cows produced five sets of twins and seven single calves, and Japanese Black cows two sets of twins and 11 single calves. The twinning rate in Holstein cows was higher (P < 0.05) than that in Japanese Black cows, 41.7% (5 of 12) vs. 15.4% (2 of 13). The calf birth weight in Holsteins was heavier (P < 0.05) than that in Japanese Black dams (24.5 kg, 33.6 kg vs. 19.3 kg, 25.5 kg for twin and single calves). The placental weight in Holstein dams calving twins was heavier than that in Holstein dams calving a single calf or in Japanese Black dams calving either twins or a single calf (6.6 kg vs. 3.5 kg, 4.6 kg or 2.8 kg). The number of placentome in Holstein dams calving twins was also higher (P < 0.05) than that in Holstein dams calving a single calf or Japanese Black dams (103.5 vs. 41.8, 67.9, 33.0). The number of placentome was approximately double in dams calving twins than that of dams calving a single calf.     

Purification and Characterization of a Substrate-Size-Recognizing Metalloendopeptidase from Streptococcus cremoris H61

During the ripening of Gouda-type cheese, two kinds of endopeptidases were found to participate in the degradation of αs1-CN(f1-23), a specific product from αs1-casein hydrolyzed by chymosin. One of the endopeptidases, lactic acid bacteria endopeptidase (LEP-II), which can recognize the size of its substrates, has already been purified and characterized (T. R. Yan, N. Azuma, S. Kaminogawa, and K. Yamauchi, Eur. J. Biochem. 163:259-265, 1987). The other endopeptidase, LEP-I, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme appeared to be monomeric, with an apparent molecular weight of 98,000, and its isoelectric point was 5.1. For the hydrolysis of αs1-CN(f1-23), the enzyme had an optimum pH and temperature of 7.0 to 7.5 and 40°C, respectively. Its activity was inhibited by such chelating agents as EDTA and 1,10-phenanthrolin, and it could be fully reactivated by Mn²⁺. Inhibitors specific for serine and thiol proteases had no effect on the protease activity. The enzyme showed a high affinity toward the Glu-Asn peptide bond of αs1-CN(f1-23) and αs1-CN(f91-100) but showed no hydrolysis activity toward αs1-CN(f1-52), αs1-CN(61-122), αs1-CN(136-196), αs1-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin. The K_m and V_max of LEP-I for αs1-CN(f1-23) were 14.2 pM and 139 U, respectively.     

Variability of histone H1 in rabbit populations

• 1.1. Perchloric acid-soluble proteins containing H1 histone as a main component were isolated from whole liver, lung and kidney of New Zealand White, Chinchilla, French Silver and Czech Spotted rabbits.
• 2.2. Proteins were resolved in a two-dimensional polyacrylamide slab gel into 4–5 spots depending on the tissue.
• 3.3. One of the histone subtypes, Hle, was found to be nonuniformly distributed within rabbit populations.
• 4.4. The prevailing fraction of animals had only a single spot of H1e (phenotype A). Approximately 10–28% of animals, depending on the breed, had two spots of H1e (e₁ and e₂; phenotype B) that slightly differed in the apparent molecular weights.
• 5.5. These two distinct gel patterns of H1e showed no tissue specificity, and the same phenotype was revealed in all tissues from the same animal.

     

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ_1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ_1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, α_S- and β-casein. Likewise, incubating Aβ_1–40 with αB-crystallin inhibited both Aβ_1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.     

HISTONE F1 OF TETRAHYMENA MACRONUCLEI : Unique Electrophoretic Properties and Phosphorylation of F1 in an Amitotic Nucleus

Histone fraction F1 has been isolated and purified from macronuclei of the ciliated protozoan, Tetrahymena pyriformis. In many respects, Tetrahymena F1 is similar to that of other organisms. It is the only Tetrahymena histone soluble in 5% perchloric acid or 5% trichloroacetic acid, has a higher molecular weight than any other Tetrahymena histone, is the histone most easily dissociated from Tetrahymena chromatin, and is susceptible to specific proteolytic cleavage. However, unlike F1 in all other organisms, Tetrahymena F1 is not the slowest-migrating histone fraction when analyzed by polyacrylamide gel electrophoresis at low pH. Tetrahymena F1 also exhibits unusual behavior in sodium dodecyl sulfate-containing polyacrylamide gels, migrating faster than calf thymus F1 at pH 10, and slower than calf thymus F1 at pH 7.6. Tetrahymena F1 was found to be highly phosphorylated in rapidly growing cells, suggesting that the relationship between cell replication and F1 phosphorylation previously observed in mammalian cells may extend to all eukaryotes. The observation that extensive F1 phosphorylation occurs in macronuclei, which divide amitotically, argues against a unique role for F1 phosphorylation in the process of chromosome condensation at mitosis.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC₅₀s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC₅₀s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.