Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

Exopolysaccharides production as affected by lactic acid bacteria and fermentation time

The aim of this work was to examine the ropiness of Lactobacillus helveticus BCRC14030, L. helveticus BCRC14076, and Streptococcus thermophilus BCRC14085, and evaluate the effect of fermentation time on exopolysaccharides (EPS) production by the ropy strain. Each of the three strains was inoculated in skim milk medium and incubated in a fermenter for 0–84 h at pH 5, 37 °C. The fermented samples, containing a net volume of 300 ml skim milk, were withdrawn at intervals of 0, 12, 16, 24, 32, 36, 48, 60, 72, and 84 h for determinations of ropy condition, EPS yield, and molecular mass. EPS with ropiness values of 11.3–21.0 mm, produced from L. helveticus BCRC14030 at 32–60 h demonstrated the ropy nature of the strain. Those EPS were composed of high molecular mass of 26,500 kDa. The highest EPS yield of 0.73 g l⁻¹ from this strain was observed (P < 0.05) at less favourable fermentation condition of 60 h. In addition, a relationship between the presence of high molecular mass and the ropiness of EPS from L. helveticus BCRC14030 was revealed.     

Short communication: Phenotypic characterization of total antioxidant activity of buffalo,goat, and sheep milk

Free radicals are reactive and unstable waste molecules produced by cells, responsible of damages and alteration on DNA, proteins, and fat. The daily intake of antioxidant compounds, acting against free radicals and their detrimental effects, is essential for human health. Milk contains several compounds with antioxidant activity, and the sum of their reducing potential blocking free radicals development is defined as total antioxidant activity (TAA). This novel trait has been described in literature both in individual and bulk cow milk, but there are no reports from other dairy species. Therefore, the present study aimed to investigate phenotypic variation of TAA in individual samples of buffalo (n = 105), goat (n = 112), and sheep (n = 198) milk. Total antioxidant activity was measured through a reference spectrophotometric method, and expressed as millimoles per liter of Trolox equivalents (TE). The greatest TAA was observed in sheep milk, averaging 7.78 mmol/L of TE and showing also the broadest phenotypic variation expressed as coefficient of variation (13.98%). Significantly lower TAA values were observed for buffalo (7.35 mmol/L of TE) and goat (6.80 mmol/L of TE) milk, with coefficients of variation of 8.18 and 8.47%, respectively. Total antioxidant activity exhibited weak correlations with milk yield and chemical composition. Phenotypic values of TAA presented in this study will be used to assess the ability of mid-infrared spectroscopy to predict this new trait and thus to collect data at the population level.     

Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece

From December 1999 to May 2000, 114 samples of pasteurized, ultrahigh temperature-treated (UHT) and concentrated milk were collected in supermarkets, whereas 52 raw milk samples from cow, sheep and goat were obtained from different milk producers all over Greece. Sample collection was repeated from December 2000 to May 2001 and concerned 54 samples of pasteurized milk, 23 samples of bulk-tank raw milk and 55 raw milk samples from cow, sheep and goat. The total number of samples analysed for aflatoxin M₁ (AFM₁) contamination by immunoaffinity column extraction and liquid chromatography was 297. In the first sampling, the incidence rates of AFM 1 contamination in pasteurized, UHT, concentrated and cow, sheep and goat raw milk were 85.4, 82.3, 93.3, 73.3, 66.7 and 40%, respectively, with only one cow raw milk and two concentrated milk samples exceeding the EU limit of 50 ng l^-1. In the second sampling, the incidence rates of AFM 1 contamination in pasteurized, bulk-tank and cow, sheep and goat raw milk were 79.6, 78.3, 64.3, 73.3 and 66.7%, respectively, with only one cow and one sheep raw milk samples exceeding the limit of 50 ng l^-1. The results suggest that the current regulatory status in Greece is effective.     

一株食源性单增李斯特菌镉、砷耐受基因的测序分析

酪蛋白磷酸肽（casein phosphopeptides,CPP）是以牛乳酪蛋白为原料,含有成簇的磷酸丝氨酸的生物活性肽。本文通过比较两种不同氮磷摩尔比（N/P）的CPP样品抑制钙离子形成沉淀的功能以及pH值、温度和CPP浓度对其的影响,并与柠檬酸和木糖醇的协同作用进行比较研究。结果表明：CPP浓度是CPP作用效果的主要影响因素,pH值对CPP影响较大,其次是柠檬酸和木糖醇的添加。温度对CPP作用效果几乎没有影响;柠檬酸和木糖醇与两种CPP之间都有一定的协同作用并且两种CPP与柠檬酸的协同作用效果接近。且浓度越高,效果越好。在抑制金属离子沉淀,pH、CPP浓度和木糖醇协同作用上CPPⅡ的作用效果较好,但温度对其两者都没有影响。两种CPP样品都具有良好的功能性质,但氮磷摩尔比（N/P）低的CPPⅡ性质更优。本试验为我国研究开发富含CPP的功能性食品以及营养保健食品提供理论基础。     

Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese

The aim of this work was to develop an assay capable of detecting adulteration of soft goat, sheep and buffalo milk cheese with bovine milk from cheaper sources. A previously developed indirect competitive ELISA had a lower sensitivity when applied to cheese, compared with milk. A sandwich ELISA was developed utilising the same monoclonal antibody in combination with a polyclonal goat anti-bovine IgG antibody. Once optimised, the ELISA was found to be highly specific. Detection limits in milk were 0.001% cows’ milk adulteration of sheep or buffalo milk, and 0.01% cows’ milk adulteration of goat milk. Detection limits in soft cheese were 0.001% in goat cheese and 0.01% in sheep or buffalo cheese. The assay was highly reproducible with both intra- and inter-assay coefficient of variation <10%. The ELISA performance makes it suitable for development as a kit for use in routine surveillance of milk and soft cheese.     

Validation of a gold standard method for iodine quantification in raw and processed milk,and its variation in different dairy species

Adequate milk consumption significantly contributes to meeting the human iodine recommended daily intake, which ranges from 70 µg/d for infants to 200 µg/d for lactating women. The fulfilment of iodine recommended daily intake is fundamental to prevent serious clinical diseases such as cretinism in infants and goiter in adults. In the present study iodine content was measured in raw and processed commercial cow milk, as well as in raw buffalo, goat, sheep, and donkey milk. Iodine extraction was based on 0.6% (vol/vol) ammonia, whereas iodine detection and quantification were carried out through an inductively coupled plasma mass spectrometer analyzer. Among processed commercial cow milk, partially skimmed pasteurized milk had the greatest iodine content (359.42 µg/kg) and raw milk the lowest (166.92 µg/kg). With regard to the other dairy species, the greatest iodine content was found in raw goat milk (575.42 µg/kg), followed by raw buffalo (229.82 µg/kg), sheep (192.64 µg/kg), and donkey milk (7.06 µg/kg). Repeatability of milk iodine content, calculated as relative standard deviation of 5 measurements within a day or operator, ranged from 0.96 to 1.84% and 0.72 to 1.16%, respectively. The overall reproducibility of milk iodine content, calculated as relative standard deviation of 45 measurements across 3 d of analyses and 3 operators, was 4.01%. These results underline the precision of the proposed analytical method for the determination of iodine content in milk.     

Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

A novel method for species identification in milk and milk-based products

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic kappa-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0.1% bovine milk mixed with buffalo milk.     

Characterization of major and trace minerals,fatty acid composition,and cholesterol content of Protected Designation of Origin cheeses

Cheese provides essential nutrients for human nutrition and health, such as minerals and fatty acids (FA). Its composition varies according to milk origin (e.g., species and breed), rearing conditions (e.g., feeding and management), and cheese-making technology (e.g., coagulation process, addition of salt, ripening period). In recent years, cheese production has increased worldwide. Italy is one of the main producers and exporters of cheese. This study aimed to describe mineral, FA, and cholesterol content of 133 samples from 18 commercial cheeses from 4 dairy species (buffalo, cow, goat, and sheep) and from 3 classes of moisture content (hard, <35% moisture; semi-hard, 35–45%; and soft, >45%). Mineral concentrations of cheese samples were determined by inductively coupled plasma optical emission spectrometry, and FA and cholesterol contents were determined by gas chromatography. Moisture and species had a significant effect on almost all traits: the highest levels of Na, Ca, and Fe were found in cheeses made from sheep milk; the greatest level of Cu was found in cow milk cheese, the lowest amount of K was found in buffalo milk cheese, and the lowest amount of Zn was found in goat cheeses. In all samples, Cr and Pb were not detected (below the level of detection). In general, total fat, protein, and minerals significantly increased when the moisture decreased. Buffalo and goat cheeses had the highest saturated FA content, and sheep cheeses showed the highest content of unsaturated and polyunsaturated FA, conjugated linoleic acid, and n-3 FA. Goat and sheep cheeses achieved higher proportions of minor FA than did cow and buffalo cheeses. Buffalo cheese exhibited the lowest cholesterol level. Our results confirm that cheese mineral content is mainly affected by the cheese-making process, whereas FA profile mainly reflects the FA composition of the source milk. This study allowed the characterization of mineral and FA composition and cholesterol content and revealed large variability among different commercial cheeses.     

High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by glmM gene: a risk of food-borne infection?

Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.     

Accelerated ripening of low fat Ras cheese by attenuated lactobacilli cells

Thirteen Ras cheese were made from 4% fat raw milk; 3% raw and heat treated; 2% raw and heat treated milks in order to study the effect of freeze-shocked or heat-shocked L. casei NIH 334 or L. helveticus CNRZ 53 on the quality of the resultant cheeses. The soluble nitrogen, soluble tyrosine, soluble tryptophan, total volatile fatty acids, titratable acidity and organoleptic evaluation scores increased as ripening period progressed, while moisture decreased. Neither strain nor the heated lactobacilli had significant effects on moisture content of cheeses, while increasing their acidity. Cheeses with freeze-shocked L. casei or L. helveticus had higher titratable acidity than cheeses in which heat-shocked cells were added. However, cheeses added L. helveticus had higher acidity than those with L. casei. Ripening indices (soluble nitrogen, soluble tyrosine, soluble tryptophan and total volatile fatty acids) and organoleptic evaluation scores had similar trends. Cheeses with attenuated lactobacilli had higher ripening indices and cheese scores than cheeses without lactobacilli. Addition of either freeze-shocked L. casei or L. helveticus yielded cheeses having higher ripening indices and organoleptic scores than cheeses made with heat-shocked lactobacilli. The best cheeses were made from 3% fat milk heated to 70 °C, and containing freeze-shocked L. helveticus followed by cheeses made from 2% fat milk heated to 75 °C and containing freeze-shocked L. helveticus.     

Yeasts from Water Buffalo Mozzarella, a traditional cheese of the Mediterranean area

Countries of the Mediterranean area are characterized by production of artisanal cheeses, obtained from goat, sheep, cow and buffalo raw milk. The numbers and species of yeasts in the different cheeses are variable, but some species are more frequently detected than others. Kluyveromyces marxianus, K. lactis with their anamorph, Candida kefir, Debaryomyces hansenii and C. famata, C. colliculosa and C. catenulata are dominant species in several cheeses. However, Saccharomyces cerevisiae is often detected in pasta filata cheeses, such as Water Buffalo Mozzarella (WBM) or Cacio Cavallo Podolico. Recently, a comprehensive study of yeasts isolated from Mozzarella cheese produced in Basilicata (Southern Italy) has been carried out. The study has focused on lactose and/or galactose fermenting species (Kluyveromyces and Saccharomyces) to evaluate their role on the functional and sensory properties of the product. End products in milk were evaluated and the biodiversity in terms of production of sulphur dioxide, higher alcohols, ethyl acetate, and acetaldehyde was studied. In particular, S. cerevisiae strains from Water Buffalo Mozzarella cheese, compared to strains isolated from different habitats, such as wine, exhibited considerable difference in the production of some volatile compounds. The diversity observed could be related to the particular microhabitat of S. cerevisiae occurring in whey cheese of water buffalo milk.     

Molecular surveillance of Toxoplasma gondii in raw milk and Artisan cheese of sheep,goat, cow and water buffalo origin

This study is aimed at investigating the molecular prevalence of Toxoplasma gondii in raw milk and cheese of different animal species (sheep, goat, cow and water buffalo) in Kayseri Province, Türkiye, to provide a preliminary assessment for contamination risk. A total of 200 milk and cheese samples were analysed by real-time PCR. Toxoplasma gondii DNA was detected in two (8%) ewes and one (4%) goat raw milk sample, while none of the cheese samples were positive. These results indicated that the presence of T. gondii DNA in raw milk samples sold in Kayseri Province might be a risk factor for public health.     

3种乳源酪蛋白粒径及胶束结构的差异性

通过纳米粒度分析仪和扫描电子显微镜,分别对水牛乳、牛乳及羊乳中的酪蛋白颗粒直径大小分布情况及酪蛋白胶束结构进行研究。结果表明：水牛乳、牛乳及羊乳中酪蛋白的粒径分布及胶束结构方面存在明显的差异。水牛乳酪蛋白平均颗粒直径为182.3nm,酪蛋白颗粒互相连接成较细长的胶束,胶束之间交联成网络状;牛乳酪蛋白平均颗粒直径为207.4nm,酪蛋白颗粒聚集成直径较大的胶束;羊乳酪蛋白平均颗粒直径为173.8nm,酪蛋白颗粒仅能够形成较短的胶束,也不能交联成网络状。     

不同哺乳动物乳中主要营养成分比较的研究进展

通过对牛乳、水牛乳、绵羊乳、山羊乳、骆驼乳和人乳中的蛋白质、脂肪、乳糖、矿物质和维生素等主要营养成分进行比较,发现山羊乳总体营养成分接近于人乳,对婴幼儿有极高的营养价值;绵羊乳和水牛乳中干物质含量较高,有利于开发成乳粉类产品;骆驼乳中含有丰富的不饱和脂肪酸,应在功能食品开发方面加以重视。     

Differentiation of closely related species by DNA hybridization

The specificity of genomic DNA probes for species differentiation by slot blot hybridization has been investigated. Experiments have been performed investigating species differentiation between monkey and human and between cattle, goat and sheep.

It is demonstrated that cross hybridization between probe and DNA sequences from closely related species is reduced by addition of unlabelled DNA from the cross hybridizing species. Quantitative species differentiation is shown possible for all species although with different detection limits. For differentiation between cattle and sheep or goat the detection limits are determined to less than 0·01% whereas the detection limits for differentiation between the closely related species sheep and goat are about 10%.     

Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.     

Nutritional Value and Technological Suitability of Milk from Various Animal Species Used for Dairy Production

Abstract: The analysis of nutritional value and selected traits of technological suitability of milk was performed on the basis of the available literature. This analysis concerned various animal species used for dairy purposes (cattle, buffalo, goats, sheep, camels, donkeys, and horses). It has been stated that a considerable diversity exists in the analyzed parameters and traits of milk, which results in various directions of milk utilization. Cow milk accounts for more than 80% of world milk production. It is the most universal raw material for processing, which is reflected in the broadest spectrum of manufactured products. Sheep and buffalo milk, regarding their high content of protein, including casein, and fat, make a very good raw material for processing, especially cheesemaking. Donkey and horse milk have the most comparable protein composition to human milk (low content of casein, lack of αs₁‐casein fraction and β‐lactoglobulin, and high content of lysozyme). Donkey milk is additionally characterized by a fatty acid profile distinctive from milk of other analyzed animal species. Camel milk also has valuable nutritional properties as it contains a high proportion of antibacterial substances and 30 times higher concentration of vitamin C in comparison to cow milk. The composition of goat milk allows using it as the raw material for dairy processing and also to some extent as a therapeutical product (low content or lack of αs₁‐casein).     

Comparative milk fatty acid analysis of different dairy species

This study compared the fatty acid (FA) profile of milk from different dairy mammals: buffalo, camel, cow, goat and yak. Data from milk samples and reports in the literature were processed using principal component analysis. The results showed that camel milk contained the lowest levels of C4:0–C12:0 and highest levels of unsaturated fatty acids. Goat milk had the highest level of C8:0–C14:0 FA. Characteristic differences were observed for yak and buffalo milk, which differed from cow milk by their higher levels of saturated fatty acids and lower levels of polyunsaturated fatty acids. It is therefore suggested that each animal species’ milk has its own specific milk FA profile.