Commercial Fermented Milk Research Articles

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.

Changes in gastric resistance of Lactobacillus casei in flavoured commercial fermented milks during refrigerated storage

This study investigated the gastric acid resistance ofLactobacillus caseiin commercial fermented milks during refrigerated storage. Samples of fermented milks (natural, strawberry, fruits of the forest, vanilla and multifruits) were obtained from three different manufacturers. In vitro gastric acid resistance (GAR) was assessed in samples upon arrival to the laboratory and after 10 and 20 days of refrigerated storage at 5 °C and 12 °C. The GAR ofL. caseiin fermented milks may increase or decrease during storage in relation to the flavour and storage conditions. The occurrence of this phenomenon in commercial fermented milks containing probiotic bacteria may induce changes in their functionality after consumption.

Keamanan Susu Fermentasi Yang Beredar Di Banda Aceh Berdasarkan Nilai Gizi dan Jumlah Bakteri Pathogen

Safety condition of commercial fermented milk that put on the market in banda aceh based on nutritive value and pathogenic bacteriaABSTRACT. Fermented milk has much value primarily for the health of digestive system. However without hygienically and sterilized treatment it can become toxic to consumers. This research was conducted to observe nutritive value, the present of pathogenic bacteria, and the safety condition of commercial fermented milk that put on the market in Banda Aceh. Samples of commercial fermented milk tested were obtained from recognized retailers in Banda Aceh. Factorial completely randomized design with six replications was applied in this research. Variety of fermented milk products (yoghurt and drink yoghurt) was the first factor to be analyzed, and the second one was expired date of the products (the date of production and the date of by expired). The amount of crude protein and lipid, pH level, and the amount of lactic acid bacteria, Coliform and Staphilococcus aureus were the parameter determined. The variety and the expired date of the products influenced the amount of crude protein and lipid, the pH level, the amount of lactic acid bacteria, Coliform and Staphilococcus aureus in the level of highly significant different (P0,01). There was also found in highly significant interaction (P0,01) between factors that influenced the amount of lactic acid bacteria and Staphilococcus aureus. On the other hand, the amount of Coliform was not influenced by those factors. The study also results in decreasing of the amount of crude protein and lipid as well as lactic acid bacteria caused by the ongoing time of storing. The longer fermented milk was stored, the lower the level of crude protein, lipid and lactic acid bacteria presented. The lipid level of fermented milk products those put on the market in Banda Aceh have been complied with the requirement standard of SNI 01-2981-1992. However, crude protein level has not achieved the requirement standard yet. Due to the amount of Staphylococcus aureus present, the fermented milk products tested, in fact, were not saved to be consumed. On the contrary, although Coliform was also present, the products were in the category of saved to be consumed.

Comparison of Bifidobacteria Selective Media for the Detection of Bifidobacteria in Korean Commercial Fermented Milk Products

This study was carried out to compare the efficacy and selectivity of TOS and BS media for enumeration of bifidobacteria in commercial fermented milk products. First, bifidobacteria was isolated from 20 fermented milk products, and all isolated bifidobacteria were identified by genomic technology as Bifidobacterium lactis. The two media significantly differed from each other with regard to the recovery of B. lactis, that is, the recovery of this organism was as much as 6 logs lower on BS medium than on TOS. When the concentration of BS solution (mixture of paromomycin sulfate, neomycin, sodium propionate, and lithium chloride) used in BS medium was reduced to 50% (BS50), a relatively high percentage recovery of bifidobacteria from pure cultures was achieved. Susceptibility tests to antibiotics and tests for selective agents for the isolated bifidobacteria and lactic acid bacteria were conducted. The BS solution inhibited some lactic acid bacteria and Bifidobacterium species, while mupirocin (MU) suppressed the growth of all tested lactic acid bacteria but not Bifidobacterium. As compared with BS50 medium, TOS with or without MU showed good bifidobacteria recovery and readily distinguishable colonies; in particular, TOS supplemented with MU had a high selectivity for bifidobacteria. In conclusion, all results suggested that TOS medium with or without MU was found to be suitable for selective enumeration of bifidobacteria from mixed cultures in fermented milk, and better in that capacity than BS medium.

Inhibition ofEscherichia coliO157:H7 in commercial and traditional fermented goat milk by activated lactoperoxidase

The lactoperoxidase (LP) system has been reported to inhibit the production of acid by lactic starter cultures and can result in the survival and growth of acid-adapted enteropathogens in LP-activated fermented milk. The aim of this study was to investigate the effect of the LP system on growth and acid production by single strains and indigenous lactic acid bacteria (LAB) and the survival of Escherichia coli O157:H7 in fermented goat’s milk. LP-activated raw and pasteurized goat’s milks were inoculated with single strains of Lactococcus sp. and Bifidobacterium longum BB536 and incubated for 24 h at 30 °C to simulate commercial milk fermentation. Madila, a traditional fermented milk product, was fermented with indigenous LAB for 5 days at 30 °C. Goat’s milk was also inoculated with E. coli O157:H7 to determine its survival during the fermentation of the LP-activated milk. The viability of LAB and E. coli O157:H7, pH and acid production were monitored during the fermentations. None of the LAB cultures tested displayed significant sensitivity to the LP system with respect to growth and acid production. However, E. coli O157:H7 was inhibited in the LP-activated milk in the commercial fermented milk and in the traditional Madila, where the counts were reduced by > 5.0 log cfu·mL−1. Therefore, the LP system could be used during the fermentation of both traditional and commercial milk processing at ambient temperatures as an additional bacteriological control to improve the quality of commercial sour milk and traditional Madila-type products.

Development and Use of tuf Gene–Based Primers for the Multiplex PCR Detection of Lactobacillus acidophilus, Lactobacillus casei Group, Lactobacillus delbrueckii, and Bifidobacterium longum in Commercial Dairy Products

PCR primers specific for the detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum were designed based on the elongation factor Tu gene (tuf). The specificity of these four primer sets were confirmed by PCR with 88 bacterial strains of Lactobacillus, Enterococcus, Bifidobacterium, and other bacterial species. Results indicated that these primer sets generated predicted PCR products of 397, 230, 202, and 161 bp for L. acidophilus, L. delbrueckii, L. casei group, and B. longum, respectively. Bacterial species other than the target organisms tested did not generate false-positive results. When these four primer sets were combined for the simultaneous detection of the lactic acid bacteria (LAB) in fermented milk products including yogurt, the LAB species listed on the labels of these products could be identified without the preenrichment step. The identification limit for each LAB strain with this multiplex PCR method was N × 103 CFU/ml in milk samples. The results of our multiplex PCR method were confirmed by PCR assay using primers based on the 16S rDNA or the 16S–23S intergenic spacer region and by biochemical tests using the API 50 CHL kit. When this multiplex PCR method was used with the determination of counts of total viable LAB and bifidobacteria, the quality of commercial fermented milk products could be assured.

Study of galactooligosaccharide composition in commercial fermented milks

A simple high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method was validated to determine the galactooligosaccharide (GOS) content of 14 commercially available fermented milks (yogurts, yogurts containing bifidobacteria, and ready-to-drink yogurts containing Lactobacillus casei) marketed in Spain. The repeatability of the method, expressed in relative standard deviation percentages, proved to be in the range 2.8–6.2%. The recovery percentages of GOS varied between 92.7% and 96.4%. The obtained results support the suitability of the method. The diversity of oligosaccharides content and their distribution profiles in commercial samples was investigated. The components identified in commercial fermented milks were d-Galp- β(1→3)- d-Gal (3-galactobiose), d-Galp- β(1→6)-Lac (6′-galactosyl-lactose), d-Galp- β(1→3)- d-Glc (3-galactosyl-glucose), and d-Galp- β(1→3)-Lac (3′-galactosyl-lactose). Individual GOS content showed a wide variation among fermented milks analysed. Commercial yogurts containing bifidobacteria showed higher amounts of GOS (0.36–0.58%) than ready-to-drink yogurts containing L. casei (0.29–0.44%) and traditional yogurts (0.22–0.25%). The total and individual GOS content remained significantly unchangeable ( P⩽0.05) after 21 d of storage at 4 °C in all analysed samples. Since GOS are recognised as prebiotic, information about their content in fermented milks would help to estimate the potential prebiotic activity of commercial products manufactured under different conditions.

Determinação da composição físico-química de produtos lácteos: estudo exploratório de comparação dos resultados obtidos por metodologia oficial e por ultra-som

O presente trabalho tem como objetivo comparar os métodos oficiais de determinação físico-química de produtos lácteos (extrato seco desengordurado, proteína, gordura e densidade) com metodologia de ultra-som. Foram analisados diferentes leites (integral e desnatado, convencional e orgânico), leites fermentados comerciais, bases lácteas para leites fermentados e soro de queijo, totalizando vinte produtos lácteos de características diferentes. O método por ultra-som é rápido e os resultados obtidos correlacionam-se àqueles das análises oficiais. Entretanto, é necessário o emprego de um perfil específico através da calibração do equipamento com base nas informações das análises oficiais. Os resultados deste trabalho exploratório sugerem a necessidade de se realizar um estudo com grande número de amostras a fim de validar as conclusões obtidas. As evidências descritas neste artigo são importantes para a indústria brasileira de produtos lácteos.

Effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages

The effect of the long-term administration of commercial fermented milk containing probiotic bacteria in the mucosal immune response and peritoneal macrophages was analyzed. BALB/c mice were fed with fermented milk for 98 consecutive days. Small and large intestines were removed for histology; IgA, CD4, CD8 cells and cytokines-producing cells were counted. The influence on the immune cells associated with bronchus and mammary glands as well as on peritoneal macrophages was also analyzed. Continuous oral administration of fermented milk increased IgA+ cells in both parts of the intestine (small and large intestine). IL-10, a regulatory cytokine, increased in the intestinal cells in most samples. TNF α, IFN γ and IL-2 producing cells were also enhanced. Values for CD4 and CD8 + cell populations in lamina propria of the intestine were increased in relation to the control throughout the assay. No modifications in the histology of intestines were observed. Long-term consumption of fermented milk enhanced intestinal mucosa immunity, mediated by IgA+ cells and by cytokine production. This improvement of gut immunity was maintained and down-regulated by cytokines such as IL-10, preventing gut inflammatory immune response. The effect of this fermented milk on mucosal sites distant to the gut, such as bronchus and mammary glands, showed that in both tissues the increase in IgA+ cells was only observed at the beginning of the continuous consumption and no modifications in the number of cytokine positive cells were found. Similar observations were found when phagocytic activity of peritoneal macrophages was measured. It was demonstrated that the most evident effect of long-term consumption of fermented milk was observed in the intestine. Immunodulatory effects and the maintenance of intestinal homeostasis without secondary effects after long-term administration of fermented milk were also observed.

Development and Validation of an HPLC/UV Method for Quantification of Bioactive Peptides in Fermented Milks

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin‐converting‐enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow‐rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1% TFA in water and solvent B was acetonitrile‐water‐trifluoracetic acid 95∶5∶0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01–1.0 mg/mL for VPP, 0.005–1.0 mg/mL for IPP, and 0.05–3.0 mg/mL for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

Acid adaptation affects the viability of Salmonella typhimurium during the lactic fermentation of skim milk and product storage

In this study, Salmonella typhimurium was acid adapted at pH 5.5 for 4 h. The viability of the acid-adapted and non-adapted cells of S. typhimurium was investigated both during the lactic fermentation of skim milk with Streptococcus thermophilus or Lactobacillus bulgaricus, and during the storage of lactic fermented milk products at 5 °C. It was found that the viable population of S. typhimurium, regardless of acid adaptation, increased in skim milk during the initial 24 h of lactic fermentation and then declined. However, the viable population of acid-adapted S. typhimurium was significantly higher ( P < .05) than that of non-adapted cell at the end of 48 h of fermentation. Acid-adapted cells of S. typhimurium were also found to have survived better than non-adapted cells in the S. thermophilus-prepared fermented milk and two commercial lactic fermented milk products. The viability of the acid-adapted and non-adapted S. typhimurium at 5 and 37 °C in cell-free fermented milks that had their pHs adjusted to 6.4 and skim milk (pH 6.4) was further investigated. Results revealed that acid adaptation, in addition to enhancing acid tolerance, reduced the susceptibility of S. typhimurium to refrigerated temperature and other detrimental factors which might be present in lactic fermented milk products. These responses all contribute to the enhanced survival of acid-adapted S. typhimurium in the lactic fermented milk products observed in the present study.

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.

Mouthfeel and flavour of fermented whey with added hydrocolloids

High-methoxy pectin (HMP), propylene glycol alginate (PGA), carboxymethyl cellulose (CMC) and xanthan gum (XG) were added separately to whey fermented by a yoghurt starter culture at concentrations that yielded a viscosity similar to that of commercial fermented milk beverages. Mouthfeel and flavour of the samples were assessed by a highly trained panel, and volatile analysis was performed by proton-transfer reaction mass spectrometry to determine the effect of hydrocolloids on the partition of flavour compounds in the headspace. CMC and HMP produced significantly higher thickness than PGA and XG; PGA caused a ‘gritty’ mouthfeel. The yoghurt aroma of the samples containing CMC and PGA was perceived as less intense compared with the control, consistent with a reduced concentration of key volatile compounds in the headspace of these samples.

Viability assessment of lactic acid bacteria in commercial dairy products stored at 4 °C using LIVE/DEAD® BacLightTM staining and conventional plate counts

SummaryThe viability of lactic acid bacteria (LAB) from commercial fermented milks was studied during storage at 4 °C. The enumeration of total viable bacteria was assessed using fluorescence microscopy. Plate counting on selective media was used to enumerate LAB. Using LIVE/DEAD® BacLightTM viability staining, it was observed that bacterial counts decreased gradually after expiry dates, the number of viable bacteria remaining above 106 bacteria g−1 for all of the products. Viable cell counts estimated by plating onto selective media were lower than those obtained by direct microscopic counting. The viability of LAB contained in acid products decreased during their storage period at 4 °C. All products contain viable LAB ranging from 108 to 109 bacteria g−1 and could be considered as probiotic, given that the recommended minimum number of probiotic bacteria in such food products is approximately 107 bacteria mL−1 product. The number of bifidobacteria in commercial fermented milks declared to contain bifidobacteria varied from 104 to 107 bacteria mL−1. This study confirms the usefulness of fluorescent techniques for a rapid and accurate evaluation of bacterial viability in probiotic products.

Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.

Note. In Vitro Viability of Bifidobacterium Strains Isolated from Commercial Dairy Products Exposed to Human Gastrointestinal Conditions

Levels of bifidobacteria contained in commercial fermented milks in Spain were determined by fluorescent techniques. The transit tolerance of probiotic bifidobacteria strains to human gastrointestinal tract (GIT) was assessed in vitro. The number of bifidobacteria in commercial fermented milks declared to contain bifidobacteria varied from 104 to 107 bacteria/m L. Viable cell counts estimated by plating onto selective media were lower than direct counts. Bifidobacteriumstrains analysed showed different survival behaviour. Viable bacteria counts decreased considerably following exposure to gastric juice. As only intrinsic acid resistant cells survive their passage through the human intestine, the selection of acid tolerant strains is necessary for the elaboration of probiotic products. Viability of dairy bifidobacteria is affected by gastrointestinal juices but the majority of tested strains survived well at gastrointestinal conditions. The reason for this may be the low number of viable bifidobacteria contained in commercial dairy products. Adaptation and survival at low pH is likely to determine the efficacy of Bifidobacterium strains both as dairy starters and probiotic microorganisms. This study confirmed the usefulness of fluorescent techniques for a rapid and accurate evaluation of bacterial viability in probiotic products.

Identification of antioxidant and ACE‐inhibitory peptides in fermented milk

AbstractThe angiotensin‐converting enzyme (ACE)‐inhibitory activity and antioxidant properties of a commercial fermented milk from Europe were evaluated. This dairy product showed moderate ACE‐inhibitory activity and ABTS•+ radical‐scavenging capacity. The peptides from most active fractions collected by reverse phase high‐performance liquid chromatography (RP‐HPLC) were sequenced by RP‐HPLC–tandem mass spectrometry. This technique allowed rapid identification of peptides included in the most active fractions, and various potentially active peptides were recognised according to previous studies of structure–activity relationship. Three of the identified sequences had previously been described as potent ACE inhibitors. The structure of some sequences substantiated the presence of peptides with ACE‐inhibitory, antioxidant and immunomodulatory activities. Copyright © 2005 Society of Chemical Industry

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) ( Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1–8 °C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10 3 cells per ml of product.

Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion.

The angiotensin converting enzyme (ACE)-inhibitory activity of several commercial fermented milks was evaluated. Most of these products showed moderate inhibitory activity, but a few exceptions were detected. The high ACE-inhibitory activity found in some cases could be related to the origin of the milk. Two of these products were subjected to an enzymatic hydrolysis process, which simulates physiological digestion, to study the influence of digestion on ACE-inhibitory activity. The activity did not significantly change or increase during simulated gastrointestinal digestion. The peptides generated from one selected product during simulated digestion were sequenced by tandem spectrometry. Most peptides found at the end of the simulated digestion were released after 30 min of incubation with the pancreatic extract. This suggests that physiological digestion promotes the formation of active peptides from the proteins present in these fermented products. The potential ACE-inhibitory activity of the identified peptides is discussed with regard to their amino acid sequences.

Viability and diversity of probiotic Lactobacillus and Bifidobacterium populations included in commercial fermented milks

A selection of commercial fermented milks was evaluated for the presence and viability of probiotic lactobacilli and bifidobacteria. Counts of Lactobacillus spp. always remained higher than 10 5 CFU ml −1, whereas the population of Bifidobacterium spp. decreased below this level in two products. All the probiotics announced on the label were present in commercial products, yet in two fermented milks one additional group of microorganisms was also found. The yogurt microorganism Streptococcus thermophilus was present in all cases, whereas Lactobacillus delbrueckii ssp. bulgaricus was only detected in two products. Bifidobacterium was the most frequently isolated group, all commercial strains being identified as Bifidobacterium animalis/ Bifidobacterium lactis by analysis of partial sequences of the 16 rRNA gene. The same technique allowed the identification of members of Lactobacillus casei (species Lactobacillus casei/ Lactobacillus paracasei/ Lactobacillus zeae) and Lactobacillus acidophilus (species L. acidophilus sensu stricto and Lactobacillus johnsonii). The analysis of macrorrestriction profiles by pulsed-field gel electrophoresis evidenced the reduced genetic variability existing among commercial Lactobacillus and Bifidobacterium strains. The combined use of macrorrestriction analysis and carbohydrate fermentation profiles enhanced the discriminatory power of the first technique for strains differentiation. None commercial Bifidobacterium strain presented the harmful β-glucuronidase activity whereas all of them displayed β-galactosidase, α-glucosidase and α-galactosidase activity.