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.     

Effect of fermentation temperature on the properties of exopolysaccharides and the acid gelation behavior for milk fermented by Streptococcus thermophilus strains DGCC7785 and St-143

We investigated the effect of fermenting milk with 2 strains (DGCC7785 and St-143) of Streptococcus thermophilus, which are known to produce different types of exopolysaccharide (EPS) structures. The yields and physical properties of these ropy EPS were monitored during the fermentation of milk at different temperatures. We wanted to understand how these types of EPS properties affected yogurt gelation. Reconstituted skim milk was fermented at 33, 39, or 45°C until pH values reached 5.2, 4.9, 4.7, and 4.5. Molar mass of ropy EPS samples was determined using size exclusion chromatography coupled with multiangle laser light scattering. Rheological properties of fermented milk gels were analyzed using small-strain dynamic oscillatory measurements. In both strains, concentrations of ropy EPS increased during fermentation and at all temperatures. Fermentation times, by both strains, were shortest at 45°C and longest at 33°C. For both strains, molar mass of ropy EPS ranged from 2 to 4 × 10⁶ g/mol during fermentation. A major proteinaceous contaminant that was co-isolated with the ropy EPS fraction by our isolation method was identified as a milk-derived phosphoglycoprotein PP3. Increase in fermentation temperature from 33 to 45°C significantly decreased the storage modulus values (from 170 to 41 Pa) for milk gelled by strain DGCC7785, whereas the gels made with St-143 had very low storage modulus values (11–17 Pa) regardless of fermentation temperatures. For both strains, the values of maximum loss tangent in the milk gels increased with fermentation temperature; the maximum loss tangent occurred at higher pH values when milk was fermented by strain DGCC7785. The specific type of EPS produced appeared to be responsible for the differences in yogurt texture rather than the concentration or molar mass of the EPS.     

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.     

ADSA Foundation Scholar Award: Possibilities and challenges of exopolysaccharide-producing lactic cultures in dairy foods

Exopolysaccharides (EPS) from lactic acid bacteria are a diverse group of polysaccharides exhibiting various functional properties. Two forms of EPS are produced by lactic acid bacteria: capsular and unattached. Capsular EPS does not cause ropiness nor does production of unattached EPS ensure ropiness. The functions of EPS in dairy products are not completely understood. This is for 2 main reasons: the major variations among exopolysaccharides even from the same group of micro-organisms, which makes it difficult to apply information from one EPS to others, and the lack of availability of techniques with the ability to observe the microstructure and distribution of the highly hydrated EPS in fermented dairy products. The introduction of relatively new microscopic techniques such as confocal scanning laser microscopy and cryo-scanning electron microscopy made it possible to directly observe the distribution of fully hydrated EPS in dairy products. Recently, EPS produced by nonropy strains have drawn the attention of the dairy industry. This is because of the ability of some nonropy strains to produce large capsular and unattached EPS that would improve the texture of dairy products without causing the undesirable slippery mouthfeel produced by the ropy strains. Factors affecting functions of EPS are their molecular characteristics and ability to interact with milk proteins. Studying the interaction between EPS and milk proteins is complex because EPS are gradually produced during fermentation, unlike polysaccharides added directly to milk to stabilize the fermented product. The concentration and possibly molecular characteristics of EPS and protein characteristics such as charge and hydrophobicity change during fermentation. Consequently, the interaction of EPS with proteins might also change during fermentation. Exopolysaccharides provide functions that benefit reduced-fat cheeses. They bind water and increase the moisture in the nonfat portion, interfere with protein-protein interactions and reduce the rigidity of the protein network, and increase viscosity of the serum phase. This review discusses the production of capsular EPS and their role in structure formation in fermented milk, the mechanism of ropiness formation, and applications of EPS-producing cultures in reduced-fat cheeses.     

Production of cured meat color in nitrite-free Harbin red sausage by Lactobacillus fermentum fermentation

Lactobacillus fermentum was substituted for nitrite to produce cured pink color in a Chinese-style sausage. Treatments included inoculations (10⁴, 10⁶, and 10⁸ CFU/g meat) followed by fermentation at 30 °C for 8 h and then at 4 °C for 16 h. Control sausage (with sodium nitrite, 60 mg/kg meat) was cured at 4 °C for 24 h without L. fermentum. The UV–Vis spectra of pigment extract from L. fermentum-treated sausage were identical to that of nitrosylmyoglobin (NO-Mb) formed in nitrite-treated control. The NO-Mb concentration and the colorimetric a^* value of sausage treated with 10⁸ CFU/g meat of L. fermentum essentially replicated those in nitrite-cured meat. Free amino acid content in sausage treated with L. fermentum was greater and the pH slightly lower compared with the nitrite-cured control sample. This study showed that L. fermentum has the potential to substitute for nitrite in the sausage production.     

葛根发酵乳发酵条件优化

以从酸奶中筛选的高产胞外多糖（EPS）的乳酸菌为出发菌株,用葛根和脱脂乳进行乳酸菌的发酵,通过单因素试验及响应面试验对葛根发酵乳发酵条件进行优化。结果表明,最优的发酵条件为发酵温度39 ℃,葡萄糖添加量1.4%,发酵时间20 h。在此优化条件下,葛根发酵乳总黄酮含量为1.91 mg/mL,EPS含量为0.155 mg/mL。     

Development of a dairy-based exopolysaccharide bioingredient

An exopolysaccharide (EPS)-producing Streptococcus thermophilus was used to develop an “all-dairy” ingredient with increased EPS content and greater functionality for dairy applications such as Mozzarella cheese. In laboratory-scale trials, milk protein hydrolysate (MPH), whey protein isolate (WPI) and whey protein hydrolysate (WPH) in reconstituted low-heat skim milk powder (LHSMP) were examined as additional dairy protein sources to increase the production of EPS. WPH supplementation resulted in the greatest EPS production. In batch fermentation, a medium based on LHSMP and WPH, combined with optimum conditions of temperature and pH, was used to obtain maximum production of EPS from S. thermophilus. EPS production was growth associated. The fermented biomass was harvested at the end of the exponential phase and freeze-dried. The reduced viable cell count and the retention of ropiness of the powder from the drying process enabled a higher level of EPS inoculation in a preliminary Mozzarella cheese manufacturing trial.     

Influence of heat impact in reconstituted skim milk on the properties of yoghurt fermented by ropy or non‐ropy starter cultures

The paper describes studies on the influence of heat impact in reconstituted skim milk on chemical and functional properties of yoghurt products. Reconstituted skim milk was heated for 20 min at 85°C, 90°C, or 95°C. Ropy (producing exopolysaccharides, EPS) or non‐ropy strains of S. thermophilus and L. delbrueckii subsp. bulgaricus were used as starter culture for yoghurt manufacture. The studies have shown that the fermentation times decreased with increasing heat impact when the ropy starter culture was used, while they remained to a far extent unchanged if the non‐ropy starter culture was applied. The lactic acid contents of the yoghurt products were in the same range when the milk was heated at 85°C or 90°C, while they were different when milk was heated at 95°C. There was a tendency visible that an increase in preheating leads to increased L (+)‐ and decreased D (–)‐lactic acid contents if the non‐ropy culture was applied. Using the ropy culture, it was vice versa. A slightly decrease in proteolysis with increasing heat impact was to be noted with both starter cultures. Concerning the relation of proteolysis to acidification, the fermentation process could be subdivided into three sections with different slopes if the non‐ropy starter culture was used, while a linear relation was found if the ropy starter culture was applied. Regarding final product characteristics it was found that the functional properties of yoghurt decreased with increasing heat impact when the ropy starter culture was applied, while they remained to a far extent unchanged when the non‐ropy starter culture was used. It can be concluded from these studies that a preheating of milk at a temperature of 85°C (20 min) is optimal in regard to final yoghurt product characteristics.     

Ropy exopolysaccharide-producing Bifidobacterium longum YS108R as a starter culture for fermented milk

Bifidobacteria are generally inferior compared to traditional starter lactic acid bacteria (LAB) in terms of fermentation properties. The results of this study showed that the use of a ropy EPS producing strain Bifidobacterium longumYS108R during fermentation improved the physicochemical properties of fermented milk, including decreased whey syneresis and increased water-holding capacity and apparent viscosity, however, another non-ropy EPS producing strain B. longum C11A10B did not exert such effects. Furthermore, we found that synergistically fermented milk with YS108R and commercial starter could reduce the production of acetic acid, a metabolite of bifidobacteria, and scored better in terms of sensory evaluation, especially with regards to mouthfeel texture. Comparative genomic analysis revealed a single mutation in the protein-tyrosine kinase gene of strain YS108R, which may relate to ropy phenotype. Our results indicate that ropy-EPS producing B. longumYS108R is suitable as an adjunct culture for the manufacture of novel fermented dairy products.     

Effect of 'ropy' strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus on rheology of stirred yogurt

The TA-TX2 Texture Analyser and the Brookfield RVT Viscometer have been used to investigate the contribution of ropiness to the texture of stirred yogurts made using ropy strains of bacteria. Back extrusion and texture profile analysis, not commonly used to quantify rheological properties of semi-solid foods, have been found useful in distinguishing the contribution of exopolysaccharides to different texture attributes (Toba et al ., 1990). Thus ropiness, a characteristic which is imparted to the product as a result of fermentation with particular polysaccharide-producing strains, contributes to 'adhesiveness', while 'firmness' and 'elasticity' are likely to be influenced more by the protein matrix of the yogurt than by secretion of the polysaccharide by the ropy strains. Effects on viscosity and ability to recover viscosity after disruption were apparent, although the contribution of ropiness was not always positive. Ropy strains increased viscosity of stirred yogurts when compared to yogurt made with non-ropy cultures. But, whilst a ropy Lactobacillus delbrueckii ssp. bulgaricus (Lb r⁺) combined with a non-ropy Streptococcus thermophilus (St r⁻) produced a viscous product which recovered its viscosity well, a yogurt made by combining both ropy strains did not recover its viscosity as well as yogurt made by combining two non-ropy cultures and lost its structure more rapidly during the destructive testing. These results show therefore that inclusion of a ropy strain will not always lead to improved texture attributes, that while ropy strains may increase viscosity they may not influence 'firmness' and lend support to the view that this latter attribute is more influenced by protein–protein interactions.     

Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage

Lactobacillus sake 2a is a bacteriocinogenic strain isolated from “lingüiça frescal”, a Brazilian sausage. The combined effect of modified-atmosphere (MA) packaging (100% CO₂ and 50% CO₂/50% N₂) and addition of L. sake 2a on inhibition of growth of Listeria monocytogenes was evaluated in “lingüiça” stored at 6 °C. By the end of the first week, the inhibition of L. monocytogenes due to MA was significant (P0.05) while the presence of L. sake 2a did not influence significantly the growth of the pathogen. After 14 days, a reduction of 1.3–1.4 log in counts of L. monocytogenes was observed in samples containing L. sake 2a only or MA packaged only, while a reduction of 3.5 log was detected in those submitted to both treatments. Results indicate that inhibition of L. monocytogenes in “lingüiça frescal” by the bacteriocinogenic L. sake 2a is enhanced by the packaging of the product in MA.     

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. Sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.     

INSTRUMENTAL TEXTURE OF SET AND STIRRED FERMENTED MILK. EFFECT OF A ROPY STRAIN OF LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS AND AN ENRICHED SUBSTRATE

Texture profile analysis (TPA) of stirred and set cultured milk were evaluated, using an exopolysaccharide producing strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2772) and a protein enriched substrate (retentate). In both cases, samples were compared respectively with a nonropy strain (NCFB 1489) and reconstituted skim milk substrate. The retentate fermented products were firmer in comparison with skim milk products, both at 10% of total solids. A loss of structure occurred when the retentate products were stirred causing a nine-fold decrease in hardness, whereas in cultured milk products the loss was of around three-fold. The main differences were found in adhesiveness and fracture characteristics. Set retentate ropy product was five times more adhesive than the nonropy one, whereas in stirred retentate and set skim milk products the difference was two-fold. No differences in adhesiveness were detected when skim milk products were stirred using either strain. The fracture force on ropy retentate samples increased after fracture while the nonropy ones showed a decrease, implying more structural breakdown in the latter. On set products, cohesiveness increased slightly due to the ropy strain. Changes in texture observed between ropy and nonropy strains can be attributed to exopolysaccharide attachment to the casein matrix being increased when the protein content is higher.     

Selection of Streptococcus thermophilus strains able to produce exopolysaccharides in milk

A collection of 139 Streptococcus thermophilus strains was genetically screened by PCR targeting some genes involved in exopolysaccharide (EPS) biosynthesis. Twenty‐nine strains were found to be PCR positive and were grown in skim milk to highlight their ropy character, that is the ability to efficiently thicken the milk cultures and minimise syneresis. Three strains producing a skim milk culture with apparent viscosities of 275 ± 14, 340 ± 21 and 510 ± 42 mPa.s, significantly higher than the average value (135 ± 87.3 mPa.s), were selected. EPS from milk cultures of these three strains was extracted and quantified from 84.4 to 209.2 mg/L.     

The occurrence of Listeria monocytogenes in cheese from a manufacturer associated with a case of listeriosis

A case of listeriosis was associated with the consumption of a soft cheese produced in England. Goats cheese and other products from the same food manufacturer were examined for the presence of Listeria over the following 11 months. Listeria monocytogenes was isolated from 16 of 25 cheese samples on retail sale, 12 of 24 cheese samples obtained directly from the factory, and from shelving within the plant. Phage-typing of 68 isolates of L. monocytogenes from cheese samples and the factory showed that 66 (97%) were indistinguishable from the strain isolated from the patient's cerebrospinal fluid and stool. L. monocytogenes was not isolated from seven goats milk or two yoghurt samples. Listeria innocua was isolated from 10 cheese samples, two of which contained no other species of Listeria. Levels of L. monocytogenes shortly after production were low (<10/g), but were higher (10⁵–10⁷ cfu/g) in six of the 16 cheese samples obtained from retail outlets. Multiplication of L. monocytogenes was demonstrated in cheeses contaminated at the factory and held at 4°C in the laboratory.     

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays

A total of 226 lactic acid bacteria (LAB) isolated from “Alheira”, a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from “Alheiras” and to select strains that could be used in “Alheira” production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of “Alheira mass” (sterilized), at 4 °C. The growth of L. innocua population was significantly suppressed in the paste of “Alheira” when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1 × 10⁷ CFU/g after 28 days of incubation).     

Influence of heat impact in reconstituted skim milk on the properties of yoghurt fermented by ropy or non-ropy starter cultures

The paper describes studies on the influence of heat impact in reconstituted skim milk on chemical and functional properties of yoghurt products. Reconstituted skim milk was heated for 20 min at 85 degrees C, 90 degrees C, or 95 degrees C. Ropy (producing exopolysaccharides, EPS) or non-ropy strains of S. thermophilus and L. delbrueckii subsp. bulgaricus were used as starter culture for yoghurt manufacture. The studies have shown that the fermentation times decreased with increasing heat impact when the ropy starter culture was used, while they remained to a far extent unchanged if the non-ropy starter culture was applied. The lactic acid contents of the yoghurt products were in the same range when the milk was heated at 85 degrees C or 90 degrees C, while they were different when milk was heated at 95 degrees C. There was a tendency visible that an increase in preheating leads to increased L(+)- and decreased D(-)-lactic acid contents if the non-ropy culture was applied. Using the ropy culture, it was vice versa. A slightly decrease in proteolysis with increasing heat impact was to be noted with both starter cultures. Concerning the relation of proteolysis to acidification, the fermentation process could be subdivided into three sections with different slopes if the non-ropy starter culture was used, while a linear relation was found if the ropy starter culture was applied. Regarding final product characteristics it was found that the functional properties of yoghurt decreased with increasing heat impact when the ropy starter culture was applied, while they remained to a far extent unchanged when the non-ropy starter culture was used. It can be concluded from these studies that a preheating of milk at a temperature of 85 degrees C (20 min) is optimal in regard to final yoghurt product characteristics.     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

Survival of ropy slime-producing lactic acid bacteria in heat processes used in the meat industry

The study reported here deals with the survival of ropy slime-producing lactic acid bacteria in two different heat processes used in the Finnish meat industry. In both processes tested, ropiness was prevented in products inoculated with the bacteria before cooking. The bacterial count in the sausages decreased during processing over 4–5 log units/g. The bacteria were evidently unable to survive the processes, and the contamination of the products apparently takes place after cooking. Heat treatment given to vacuum-packed grill sausages after packing could delay the formation of ropy slime.     

肠膜明串珠菌HDE1的分离鉴定及其胞外多糖抗氧化和牛奶凝结特性研究

为了拓展产乳酸菌胞外多糖的来源,获得来源明确、产量稳定、具有优良生物学特性的乳酸菌胞外多糖。本研究从自制橘子发酵液中利用产黏菌落法分离筛选得到一株高产胞外多糖的乳酸菌,综合形态学观察及16S rDNA序列分析结果、API 50 CHL试验,对其进行鉴定,利用抗氧化及牛奶凝结试验研究了该乳酸菌胞外多糖的抗氧化及牛奶凝结等特性。结果表明,本研究获得了一株肠膜明串珠菌（Leuconostoc mesenteroides）,该菌株16S rDNA序列片段长度为1444 bp,GenBank登录号为OM302141。菌株HDE1胞外多糖的总糖、蛋白质、糖醛酸含量分别为41.73%±1.74%、0.29%±0.03%和7.69%±0.42%。该胞外多糖在浓度为3 mg/mL时展现出良好的抗氧化活性,当胞外多糖浓度为5 mg/mL时DPPH自由基清除能力达到50.00%±0.05%,ABTS+自由基清除能力达到40.00%±0.02%,H₂O₂-自由基清除能力超过了50%,羟基自由基清除能力达到49.96%±0.03%,胞外多糖的总还原力为38.82%±0.09%。牛奶凝结研究结果表明,HDE1在36 h能够使添加3%（w/v）蔗糖的脱脂牛奶完全凝结。这些结果表明菌株HDE1胞外多糖具有良好的抗氧化和牛奶凝结特性,在食品、医药及益生领域展现出良好应用潜力。