Evaluation of culture media for selective enumeration of probiotic strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese starters

A series of culture media were evaluated for selective enumeration of 10 commercial probiotic cultures. The media M17 and MRS 5.2 were most suitable to enumerate the yoghurt starters Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. For the enumeration of probiotic Lb. acidophilus cultures, the MRS-clindamycin medium was found to be most optimal. Selective recovery results of the tested Bifidobacterium sp. were not uniform but indicated strain-to-strain variations depending on whether NPLN or MRS-LP were used. For the enumeration of probiotic cultures of Lb. rhamnosus and Lb. paracasei, LC medium (for yoghurt products) and MRS-AC medium (for cheese products) are recommended. In conclusion, our data indicate that the choice of culture medium and methodology for selective enumeration of commercial probiotic strains in combination with starters depends strongly on the product matrix, the target group and the taxonomic diversity of the bacterial background flora in the product.     

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non-selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 °C or 42 °C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37 °C or 42 °C, aerobiosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37 °C or 42 °C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 °C or 42 °C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 °C, anaerobiosis, L. acidophilus: LC medium, 42 °C, aerobiosis or anaerobiosis and L. casei: LP-MRS, 42 °C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P ≤ 0.05).     

Probiotic Cheddar cheese: Influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and organic acid profiles

Bifidobacterium longum 1941, Bifidobacterium animalis subsp. lactis LAFTI^®B94 (B94), Lactobacillus casei 279, Lb. casei LAFTI^®L26 (L26), Lactobacillus acidophilus 4962 or Lb. acidophilus LAFTI^®L10 (L10) were used as an adjunct in the production of Cheddar cheeses which were ripened for 24 wk at 4 and 8 °C. Effects of ripening temperatures on survival of starter lactococci and probiotic microorganisms, pH and composition of cheeses and production of organic acids were examined. The counts of starter lactococci in cheeses produced with B. animalis B94, Lb. casei L26 or Lb. acidophilus 4962 ripened at 8 °C were significantly lower than those ripened at 4 °C (P < 0.05) at 24 wk. Probiotic microorganisms remained viable (>7.50 log₁₀ CFU/g) at the end of 24 wk and their viability was not affected by the ripening temperatures. There were significant effects of the type of probiotic microorganisms used, ripening time, ripening temperatures and their interactions on the concentration of lactic and acetic acids in the cheeses (P < 0.05). The acetic acid concentration in cheeses made with Bifidobacterium sp. or Lb. casei sp. was significantly higher than that of the control cheese (P < 0.05). Citric, propionic and succinic acids contents of the cheeses were not significantly affected by the type of probiotic microorganisms or ripening temperatures (P > 0.05).     

Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.     

Survival of micro‐organisms and organic acid profile of probiotic Cheddar cheese from buffalo milk during accelerated ripening

The study aimed to assess the impact of ripening at elevated temperatures on the survival of probiotic micro‐organisms and production of organic acids in Cheddar cheese. Cheese was manufactured from buffalo milk using lactococci starters along with different probiotic bacteria (Lactobacillus acidophilus LA‐5, Bifidobacterium bifidum Bb‐11 and Bifidobacterium longum BB536) as adjunct cultures. The cheeses were ripened at 4–6 °C or 12–14 °C for 180 days and examined for composition, organic acids and microbial survival. The production of organic acids was accelerated at 12–14 °C when compared to normal ripening temperatures. The probiotic bacteria increased production of lactic and acetic acids, compared to cheese made with lactococci alone. The survival of the mesophilic starters was significantly (P < 0.05) reduced in all the cheese samples ripened at the higher temperature. However, the probiotic bacteria remained viable (>7.0 log₁₀ cfu/g) throughout the 180 days of ripening, irrespective of temperature. It was concluded that Cheddar containing additional probiotic cultures can effectively be ripened at elevated temperatures without any adverse effects.     

Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media

Various selective media for enumerating probiotic and cheese cultures were screened, with 6 media then used to study survival of probiotic bacteria in full-fat and low-fat Cheddar cheese. Commercial strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, or Bifidobacterium lactis were added as probiotic adjuncts. The selective media, designed to promote growth of certain lactic acid bacteria (LAB) over others or to differentiate between LAB, were used to detect individual LAB types during cheese storage. Commercial strains of Lactococcus, Lactobacillus, and Bifidobacterium spp. were initially screened on the 6 selective media along with nonstarter LAB (NSLAB) isolates. The microbial flora of the cheeses was analyzed during 9 mo of storage at 6°C. Many NSLAB were able to grow on media presumed selective for Lactococcus, Bifidobacterium spp., or Lb. acidophilus, which became apparent after 90 d of cheese storage, Between 90 and 120 d of storage, bacterial counts changed on media selective for Bifidobacterium spp., suggesting growth of NSLAB. Appearance of NSLAB on Lb. casei selective media [de man, Rogosa, and Sharpe (MRS) + vancomycin] occurred sooner (30 d) in low-fat cheese than in full-fat control cheeses. Differentiation between NSLAB and Lactococcus was achieved by counting after 18 to 24 h when the NSLAB colonies were only pinpoint in size. Growth of NSLAB on the various selective media during aging means that probiotic adjunct cultures added during cheesemaking can only be enumerated with confidence on selective media for up to 3 or 4 mo. After this time, growth of NSLAB obfuscates enumeration of probiotic adjuncts. When adjunct Lb. casei or Lb. paracasei cultures are added during cheesemaking, they appear to remain at high numbers for a long time (9 mo) when counted on MRS + vancomycin medium, but a reasonable probability exists that they have been overtaken by NSLAB, which also grow readily on this medium. Enumeration using multiple selective media can provide insight into whether it is the actual adjunct culture or a NSLAB strain that is being enumerated.     

Incorporation of Lactobacillus acidophilus in Minas fresh cheese and its implications for textural and sensorial properties during storage

The effect of Lactobacillus acidophilus on instrumental texture profile and related properties of Minas fresh cheese during storage at 5 °C and on sensory performance was investigated. Four cheese-making trials were prepared, two supplemented with a mesophilic type O culture (T1, T2) and two with lactic acid (T3, T4). L. acidophilus was added in T2 and T3. The viability of L. acidophilus, instrumental texture profile analysis and related properties were monitored during storage for up to 21 days. Probiotic cheeses T3 were firmer by the end of storage, due to higher values of pH and hardness. Differences detected were attributed to the starter, rather than to L. acidophilus. Viability of L. acidophilus during storage ranged from 6.04 to 6.93 for T2 and from 5.46 to 6.53 log cfu g⁻¹ for T3, which performed better in sensory evaluation. Minas fresh cheese is a suitable food system for the delivery of L. acidophilus.     

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23 s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.     

Suitability of whey and buttermilk for the growth and frozen storage of probiotic lactobacilli

The growth of six probiotic commercial strains of lactobacilli was assessed in reconstituted dried whey and buttermilk supplemented with yeast extract, meat peptone, soy peptone, tryptone or casein acid hydrolysate at 0.3%, 0.6% or 1%. The addition of 1% glucose was also tested. Growth and acidification kinetics were determined at 37°C using MRS broth and a commercial culture medium as references. The suitability of whey and buttermilk as cryoprotectants at –20°C and –70°C was also assessed. Whey and buttermilk with 0.3% yeast extract were chosen for the growth of probiotic lactobacilli, since no satisfactory growth was observed without an external nitrogen source, whereas glucose did not improve the growth of any of the strains assayed. In general, buttermilk performed as satisfactorily as the reference media. The effectiveness of these media as cryoprotectants was strain dependent: skimmed milk and whey were the most suitable ones, especially for long-term storage at –20°C. However, at –70°C, no significant differences were observed between the culture media assessed. The use of whey or buttermilk as culture media for the production of probiotic lactic acid bacteria and for their cryopreservation implies a novel use of these low-cost products, offering an alternative way of utilizing the by-products of the dairy industry, helping to minimize their negative impact on the environment.     

Acidification rates of probiotic bacteria in Minas frescal cheese whey

The acidification rates of Lactobacillus delbrueckii subsp. bulgaricus (Lb), Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), and Bifidobacterium animalis subsp. lactis (Bl) in co-culture with Streptococcus thermophilus (St) were studied in Minas frescal cheese whey. Effects of the co-culture composition and the final pH values on the kinetic parameters of acidification, post-acidification and counts of health promoting micro-organisms were also studied. Fermentation time to reach pH 4.5 was longer when St-Lr co-culture was used, while St-Lb had the shortest fermentation time when compared with the other co-culture combinations. All products showed development of acidity during the storage period and lowest values had been observed employing St-Bl co-culture. The technological interest of using M. frescal cheese whey for the production of a probiotic lactic beverage is discussed in this article.     

Assessment of the probiotic potential of lactic acid bacteria isolated from Minas artisanal cheese produced in the Campo das Vertentes region,Brazil

The microbiota of the Brazilian Minas artisanal cheese, made from raw milk, is not well known and may include probiotic bacteria. This study aimed to assess the in vitro and in vivo probiotic properties of the lactic acid bacteria isolated from these cheeses. Thirty‐six samples of the Lactobacillus/Pediococcus group were selected for in vitro investigation. Pediococcus acidilactici (PA2) and Lactobacillus plantarum (LP4) showed the best results and were tested for their ability to protect Salmonella Typhimurium orally infected mice. LP4 showed better probiotic potential than PA2 and allowed higher values of weight gain (P < 0.05). Thus, Lb. plantarumLP4 may have potential use as a probiotic in foods after future technological screening.     

The influence of probiotic bacteria on the properties of Iranian white cheese

The viability of Bifidobacterium animalis (ATCC 25527) and Lactobacillus rhamnosus (ATCC 7469), study of interaction between probiotic and starter and their effect on the properties of Iranian white cheese were investigated during 60 days of ripening. The results indicated that probiotics did not exert any effect on the growth of commercial starters, but starters showed a synergistic effect on the growth of probiotics, and probiotics survived above the recommended level for the therapeutic minimum (10⁶–10⁷ cfu/g) after 60 days. Addition of probiotic adjunct did not alter the chemical composition except for moisture and protein. Also, the highest pH value was in probiotic cheeses without starter.     

A probiotic soy-based innovative product as an alternative to petit-suisse cheese

The present study aimed to develop a probiotic soy-based product similar to petit-suisse cheese and to evaluate its perspectives regarding potential for consumer health benefits, sensory acceptability, and instrumental texture during storage. Three different trials were studied: MP (milk-based petit-suisse – control); MSP (mixed product with milk cream and soy); SP (soymilk-based product). The formulations were produced with an ABT culture, containing Lactobacillus acidophilus La-5, Bifidobacterium animalis Bb-12, and the starter Streptococcus thermophilus and stored at 4 °C for up to 28 days. Bb-12 viability remained always above 8 log cfu g⁻¹ for all trials, whereas viability of La-5 was satisfactory at the end of storage for MP (7.56 log cfu g⁻¹) and MSP (6.49 log cfu g⁻¹), but only up to 21 days (6.84 log cfu g⁻¹) for SP. The pH remained stable and was lower for MSP (p < 0.05), whereas instrumental hardness and gumminess increased in soy-based products (MSP and SP) and decreased in the control (MP). SP had the highest sensory score means (6.4) on day 21, being sensorially attractive to provide consumers with a functional food without dairy ingredients and with high viability of the probiotic microorganisms.     

Enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese: An evaluation of Baird-Parker agar,Rabbit Plasma Fibrinogen agar and the Petrifilm™ Staph Express count system

Staphylococcus spp. are microorganisms that are naturally present in milk and dairy products and are often associated with food-borne diseases outbreaks due to the ability of some strains to produce thermostable enterotoxins. This ability is usually associated with coagulase and thermonuclease production, characteristics that are considered in the microbiological analyses for the control of such microorganisms. The objective of this study was to evaluate the culture media and the methodologies used for the enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese. Samples of artificially contaminated milk (with coagulase-positive Staphylococcus reference strains) and samples of naturally contaminated raw milk and cheese were submitted for enumeration in Baird-Parker agar (BP), Rabbit Plasma Fibrinogen agar (RPFA) and in the Petrifilm™ Staph Express count system (STX). No significant differences (P > 0.05) were observed between the mean counts obtained in all of the evaluated culture media. RPFA and STX had good correlation indices between the total and typical colony counts as well as with coagulase and the thermonuclease-positive colony counts. Thus, there is a better association between coagulase and thermonuclease production to typical colony morphology developed on these culture media, leading to more accurate and reliable results than with BP, which demonstrated lower correlation indices between these counts.     

Lipolysis in probiotic and synbiotic cheese: The influence of probiotic bacteria, prebiotic compounds and ripening time on free fatty acid profiles

The influence of probiotic bacteria (Lactobacillus casei-01, Bifidobacterium lactis B94), prebiotic compounds (FOS and inulin) and ripening time (0-60 days) on the free fatty acid (FFA) profile of cheese, with special emphasis on the conjugated linoleic acid (CLA) content, was investigated. After 60 days of ripening, 10⁹-10¹⁰ cfu g⁻¹ cheese were recorded in both probiotic and synbiotic cheeses, despite harsh conditions of low pH values (4.1-5.1) and low moisture content (<30%, w/w). Increases in total FFA and CLA were observed throughout the ripening period, especially in synbiotic cheeses containing FOS and inulin (50:50) inoculated with B. lactis B94. The addition of FOS alone or combined with inulin did not significantly affect probiotic strain growth and viability during the ripening period; however, the advantage of the addition of prebiotic compounds in probiotic cheese manufacture is that it may allow the production of cheeses with improved performance as far as functional CLA compounds are concerned, as well as an improved nutritional quality reflected in a lower atherogenicity index.     

The effects of probiotic cultures on the organic acid content,texture profile and sensory attributes of Tulum cheese

The aim of this study was to evaluate the effects of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis on the organic acid, texture profile and sensory attributes of Tulum cheese. The acidity of sample increased throughout the storage. Storage time influenced the organic acid content of samples (P < 0.0001). Lightness decreased while redness increased (P < 0.05). Cohesiveness decreased during storage, whereas the values of all other Texture Profile Analyses (TPA) parameters increased. The samples produced with L. acidophilus had the highest texture and acceptability ratings, whereas samples produced with B. animalis subsp. lactis had the highest flavour score.     

Evolution and distribution of phospholipids in cheese and whey during the manufacturing of fresh cheese from cows' milk

In this study, we report on the evolution of phospholipid contents and their distribution in cheese and whey, during the production of fresh cheese. We found that the phospholipid content in the total mass of whey and fresh cheese together was greater than the content of phospholipids in the starting milk used to make them. This increase was probably due to bacterial growth during fermentation and the consequent synthesis of phospholipids to generate cell membranes. In the coagulation process, ≈20% of the phospholipids were drained in the whey. Based on this result, we conclude that whey might be an interesting source of phospholipids, mainly phosphatidylethanolamine and sphingomyelin, for the food industry.     

The effect of direct acidification on the microbiological,physicochemical and sensory properties of probiotic Minas Frescal cheese

Minas Frescal cheeses produced with the addition of the probiotic culture Bifidobacterium Bb‐12 and without (C1) or with (C2) lactic acid were evaluated in relation to the microbiological, physicochemical and sensory properties. After 28 days of storage, the cheeses without lactic acid showed lower moisture and pH, in addition to higher acidity and syneresis. This behaviour influenced the texture profile of the cheeses, making them harder and chewier. The colour attributes L* and b* diminished during the storage. The majority of the consumers classified the cheeses as having good acceptability and they would buy this type of functional food.     

Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts

Hard cheeses (Kefalotyri-like) were manufactured from caprine milk with yoghurt as a starter (A), and with its partial replacement with the probiotic adjuncts Lactobacillus rhamnosus LC 705 (B) and/or Lactobacillus paracasei ssp. paracasei DC 412 (C). Both adjuncts retarded the growth of enterococci, and the environment in cheese B did not favour the recovery of lactic acid bacteria (LAB) on Rogosa agar. However, better recovery of the LAB population on M17 agar from cheeses B and C made with adjuncts was recorded early in ripening, and this was accompanied by a greater decrease in pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein demonstrated that cheese C, made with Lb. paracasei ssp. paracasei as adjunct, is a better vehicle for delivery of live probiotic cells (10 ⁷   cfu/g) to the gastrointestinal tract than cheese B, made with Lb. rhamnosus ; the latter did not belong to the predominant microflora of one out of the two B cheeses. Urea-PAGE electrophoresis results indicated that adjunct lactobacilli enhanced the degradation of both α _S -casein (α _S -CN) and β-casein (β-CN). In the fresh cheese, hydrolysis of α _S -CN was more rapid than β-CN, and the free amino acid content of B and C was higher than in A. Lipolysis products were also higher in B and C than in A as ripening progressed, and the organoleptic characteristics of these cheeses resulted in higher scores, in the order C > B > A. Thus, making Kefalotyri-like cheese from caprine milk with probiotic lactobacilli, particularly Lb. paracasei ssp. paracasei, as adjunct can be considered an effective way of producing a cheese with a large number of probiotic cells.     

Caprine cheese with probiotic strains: the effects of ripening temperature and relative humidity on proteolysis and lipolysis

 The effects of ripening temperature, relative humidity and time on chemical and textural characteristics of a 'probiotic' goat's milk cheese were examined. The experimental layout followed a 2³ factorial design, with all possible combinations of 5  °C and 10  °C (ripening temperature), 85% and 95% (ripening relative humidity) and 1 day and 70 days (ripening time). All proteolytic indices measured (water-soluble nitrogen, trichloroacetic acid-soluble nitrogen and phosphotungstic acid-soluble nitrogen) were enhanced with increased ripening temperature to a greater extent than with increased ripening relative humidity; the increase in phosphotungstic acid-soluble nitrogen was the most significant. Free fatty acid concentrations in cheeses were not influenced by ripening relative humidity but increased with ripening temperature and time. A higher ripening temperature and a lower relative humidity gave rise to firmer cheeses. Postulated empirical models have provided a good fit to the experimental data set generated; such models were able to predict a decrease of 25 days in ripening time with no impairment of either proteolytic or lipolytic indices if a cheese were to be ripened at 10  °C (rather than 5  °C) and 95% relative humidity. Received: 23 March 1998