Evaluation of cheddar cheese as a food carrier for delivery of a probiotic strain to the gastrointestinal tract.

Cheddar cheese was evaluated as a food carrier for the delivery of viable microorganisms of Enterococcus faecium (Fargo 688; Quest Int., Naarden, The Netherlands) to the gastrointestinal tract. This strain had previously been shown to possess properties required of a probiotic microorganism including the ability to relieve irritable bowel syndrome. The strain was found to survive to high numbers in Cheddar cheese during ripening at 8 degrees C for 15 mo (4 x 10(8) cfu/g) and in yogurt during storage at 4 degrees C for 21 d (4 x 10(7) cfu/g). In an in vitro model system, Cheddar cheese was found to have a greater protective effect than yogurt upon exposure of the probiotic culture to porcine gastric juice at pH 2. Subsequently, a feeding trial involving 8 pigs per group was performed in which a rifampicin-resistant variant of the probiotic strain was fed for 21 d at a mean daily intake of 1.3 x 10(10) cfu/d from Cheddar cheese or 3.7 x 10(9) cfu/d from yogurt. During the feeding period, Cheddar cheese yielded a significantly higher mean fecal probiotic count (2 x 10(6) cfu/g of feces) than did yogurt (5.2 x 10(5) cfu/g of feces). These data indicate that mature Cheddar cheese compares very favorably with fresh yogurt as a delivery system for viable probiotic microorganisms to the gastrointestinal tract.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Viability of commercial probiotic cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in cheddar cheese

Six batches of cheddar cheese were manufactured containing different combinations of commercially available probiotic cultures from three suppliers. Duplicate cheeses contained the organisms of each supplier, a Bifidobacterium spp. (each supplier), a Lactobacillus acidophilus (2 suppliers), and either Lactobacillus casei, Lactobacillus paracasei, or Lactobacillus rhamnosus. Using selective media, the different strains were assessed for viability during cheddar cheese maturation over 32 weeks. The Bifidobacterium sp. remained at high numbers with the three strains being present in cheese at 4 x 10(7), 1.4 x 10(8), and 5 x 10(8) CFU/g after 32 weeks. Similarly the L. casei (2 x 10(7) CFU/g), L. paracasei (1.6 x 10(7) CFU/g), and L. rhamnosus (9 x 10(8) CFU/g) strains survived well; however, the L. acidophilus strains performed poorly with both decreasing in a similar manner to be present at 3.6 x 10(3) CFU/g and 4.9 x 10(3) CFU/g after 32 weeks. This study indicates that cheddar cheese is a good vehicle for a variety of commercial probiotics but survival of L. acidophilus strains will need to be improved.     

Survival of Listeria monocytogenes in vanilla-flavored soy and dairy products stored at 8 degrees C

The survival of Listeria monocytogenes V37 in vanilla-flavored yogurt (low-fat and nonfat) and soy milk (low-fat and Plus) stored at 8 degrees C for 31 days was investigated. Commercial samples of yogurt and soy milk were used. These samples were inoculated with either 10(4) or 10(7) CFU of L. monocytogenes per ml. Sampling was carried out every 3 to 4 days initially and was then carried out weekly, for a total storage time of 31 days. Each time a sample was collected, the pH of the sample was measured. After 31 days, low-fat plain, low-fat vanilla, and nonfat plain yogurt samples inoculated with 10(4) CFU/ml showed 2.5-log reductions in viable cell populations, and nonfat vanilla yogurt showed a 3.5-log reduction. For yogurt inoculated with 10(7) CFU/ml, reductions of 2.5 log CFU/ml were observed for plain low-fat and nonfat yogurts, and reductions of 5 log CFU/ml were observed for vanilla-flavored low-fat and nonfat yogurts. In vanilla-flavored and plain low-fat and Plus soy milk samples, cell counts increased from 10(4) and 10(7) CFU/ml to 10(9) CFU/ml at 7 and 3 days of storage, respectively, at 8 degrees C. Coagulation in soy milk samples was observed when the cell population reached 10(9) CFU/ml. In soy milk, the L. monocytogenes population did not change for up to 31 days. Vanillin had an inhibitory effect on L. monocytogenes in yogurt but not in soy milk.     

Behavior of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses manufactured from raw milk

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.     

Accelerated Maturation of Cheddar Cheese: Microbiology of Cheeses Supplemented with Lactobacillus casei subsp. casei L2A

Growth of lactic acid bacteria (LAB) and lactobacilli was studied in Cheddar cheeses supplemented with live and heat-shocked Lactobacillus casei subsp. casei L2A and with Neutrase® to accelerate maturation. Bacterial counts of treated cheeses rapidly reached maximal values within 1 wk, whereas the control cheese reached comparable values only after 2 mo. Addition of 1.0% heat-shocked lactobacilli led to an excellent quality Cheddar cheese with a 50% increase in flavor development, as determined by sensory evaluation, compared to control cheese. Addition of Neutrase (1 × 10^-5 AU/g cheese) permitted a gain of an additional 10% while addition of higher concentrations (2 and 4 × 10^-5 AU/g cheese) resulted in undesirable bitterness.     

Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk.

The ability of Salmonella Enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10(4) CFU/ml of a luminescent strain of Salmonella Enteritidis (lux) and 10(8) PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella Enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella Enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella Enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella Enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella Enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella Enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10(3) CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk.     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.     

Optimal growth of Lactobacillus casei in a Cheddar cheese ripening model system requires exogenous fatty acids

Flavor development in ripening Cheddar cheese depends on complex microbial and biochemical processes that are difficult to study in natural cheese. Thus, our group has developed Cheddar cheese extract (CCE) as a model system to study these processes. In previous work, we found that CCE supported growth of Lactobacillus casei, one of the most prominent nonstarter lactic acid bacteria (NSLAB) species found in ripening Cheddar cheese, to a final cell density of 10(8) cfu/mL at 37°C. However, when similar growth experiments were performed at 8°C in CCE derived from 4-mo-old cheese (4mCCE), the final cell densities obtained were only about 10(6) cfu/mL, which is at the lower end of the range of the NSLAB population expected in ripening Cheddar cheese. Here, we report that addition of Tween 80 to CCE resulted in a significant increase in the final cell density of L. casei during growth at 8°C and produced concomitant changes in cytoplasmic membrane fatty acid (CMFA) composition. Although the effect was not as dramatic, addition of milk fat or a monoacylglycerol (MAG) mixture based on the MAG profile of milk fat to 4mCCE also led to an increased final cell density of L. casei in CCE at 8°C and changes in CMFA composition. These observations suggest that optimal growth of L. casei in CCE at low temperature requires supplementation with a source of fatty acids (FA). We hypothesize that L. casei incorporates environmental FA into its CMFA, thereby reducing its energy requirement for growth. The exogenous FA may then be modified or supplemented with FA from de novo synthesis to arrive at a CMFA composition that yields the functionality (i.e., viscosity) required for growth in specific conditions. Additional studies utilizing the CCE model to investigate microbial contributions to cheese ripening should be conducted in CCE supplemented with 1% milk fat.     

Inactivation of Listeria monocytogenes on Beef Bologna and Cheddar Cheese Using Polyvinyl-idene Chloride Films Containing Sorbic Acid

ABSTRACT: Polyvinylidene chloride (PVDC, Saran^R F-310) films containing sorbic acid (0%, 1.5%, and 3.0% w/v) were prepared with use of a solvent-casting method and were then placed between slices of commercially produced beef bologna that were previously surface-inoculated with L. monocytogenes at 10³ or 10⁵ CFU/g. In addition, cubes of commercial Cheddar cheese were surface-inoculated to contain 10³ or 10⁵ Listeria monocytogenes colony-forming units (CFU) /g and then wrapped with the sorbic acid-containing films. Films containing 1.5% and 3.0% (w/ v) sorbic acid prevented growth of L. monocytogenes on bologna slices with populations as much as 7.1 logs lower after 28 d of storage at 4 °C compared with the sorbic acid-free controls. In contrast, numbers of Listeria remained relatively stable on Cheddar cheese with populations decreasing < 1.3 logs after 35 d of storage. With use of the sorbic acid-containing films, common spoilage organisms were also inhibited on both products. After 28 d of contact with bologna and Cheddar cheese, these films retained 7% and 60% of their original sorbic acid content, respectively, with the control film retaining 85% of its original sorbic acid content. Given these findings, sorbic acid-containing films may be useful in enhancing the safety and shelf-life of ready-to-eat delicatessen products.     

益生性植物乳杆菌对切达干酪挥发性风味形成的影响

将分离自西藏灵菇的益生性植物乳杆菌1-2通过在杀菌乳中添加活菌数8.0、9.0（lg（CFU/mL））和在排乳清后添加于凝乳块8.0（lg（CFU/g））的方式分别加入到切达干酪中,考察植物乳杆菌活菌数量、添加方式和成熟时间对干酪挥发性风味物质组成的影响。利用固相微萃取和气相色谱-质谱联用技术检测出对照组干酪的风味物质26种,益生菌干酪组风味物质30种,添加植物乳杆菌1-2可产生乙苯、十二烷、己醇和丙酮4种挥发性风味物质。成熟时间对干酪风味的影响最大,随成熟时间的延长,益生菌干酪组中苯含量显著增加,而对照组干酪在成熟12周时才检测到苯。益生菌添加量和添加方式对干酪挥发性风味的影响相似,丁酸受益生菌活菌数和添加方式的影响最大,益生菌干酪组成熟12周时,丁酸含量最高达对照组的3.96倍（P＜0.05）。在杀菌乳中添加益生菌活菌数8.0（lg（CFU/mL））组和9.0（lg（CFU/mL））组干酪中挥发性风味物质含量有显著差异,但在杀菌乳中添加高活菌数9.0（lg（CFU/mL））和在排乳清后添加低活菌数8.0（lg（CFU/g））于凝乳块中对干酪挥发性风味的形成具有相似的影响。本研究结果为改进益生菌干酪的加工工艺和风味品质提供了实验依据。     

Nonspecific Enzyme-Linked lmmunosorbent Assay for Molds in Foods

A nonspecific enzyme-linked immunosorbent assay (ELISA) was developed to detect molds in foods by producing an antibody to a mixture of six common molds, Aspergillus versicolor, Cladosporium herbarum, Fusarium poae, Geotrichum candidum, Mucor circinelloides, and Penicillium chrysogenum. This antibody recognized these mold genera plus 10 others but not yeasts. Mold antigens added into Cheddar and cottage cheeses, fruit juices, nonfat dry milk, raisins, and yogurt could be detected. Molds (10² spores/g) inoculated into cottage cheese and yogurt and allowed to grow at 7 or 22°C were detected at 10³ CFU/g. A nonspecific ELISA could be developed to detect general mold contamination of foods.     

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.     

Antioxidant activity of Cheddar cheeses at different stages of ripening

The aim of the study was to evaluate the changes in the antioxidant properties of Cheddar cheese at different stages of ripening using different assays: 2, 2'-azinobis (3 ethyl benzothiazoline)-6-sulphonic acid, 2, 2-diphenyl 1, picryl hydrazyl and superoxide radical scavenging activity. Cheddar cheese was prepared with Lactobacillus casei ssp. casei 300 and Lactobacillus paracasei ssp. paracasei 22 and without adjunct cultures. The antioxidant activity of water-soluble extracts of Cheddar cheese was dependent on the ripening period. The changes in the antioxidant activity were related to the rate of formation of soluble peptides (proteolysis) in all the samples of cheeses up to fourth month of ripening.     

Autochthonous adjunct culture of Limosilactobacillus mucosae CNPC007 improved the techno-functional,physicochemical, and sensory properties of goat milk Greek-style yogurt

We evaluated the performance of Limosilactobacillus mucosae CNPC007 as an autochthonous adjunct culture in the production of goat milk Greek-style yogurt. The techno-functional, physicochemical, and sensory characteristics of the control yogurt (containing only starter culture, CY) and the probiotic yogurt (with the probiotic strain added, PY) were assessed during 28 d of refrigerated storage. Furthermore, we determined the survival of the strain throughout the gastrointestinal tract under simulated conditions. The PY yogurt had a lower extent of proteolysis index and a higher depth of proteolysis index. These results indicate that the proteolytic enzymes of L. mucosae may have a possible action in PY. The PY formulation exhibited viscosity almost 1.5 times as high as CY over the refrigeration period, probably due to higher production of exopolysaccharides by the probiotic strain, which directly interferes with the microstructure, texture, and viscosity of the product. The PY formulation received higher scores for color, flavor, and global acceptance at 1 d of storage and higher texture scores at 28 d. The counts of L. mucosae remained high (>7 log cfu/g and >8.5 log cfu/g) throughout mouth-ileum digestion and storage, respectively, in PY. The autochthonous adjunct culture of L. mucosae CNPC007 can be used for production of a novel potentially probiotic goat yogurt without negatively affecting the general characteristics of the product quality, adding value associated with maintaining its functional potential.     

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.     

Manufacture of low-fat Cheddar cheese by exopolysaccharide-producing Lactobacillus plantarum JLK0142 and its functional properties

This study aimed to evaluate the effect of exopolysaccharide (EPS)-producing Lactobacillus plantarum JLK0142 on the ripening characteristics and in vitro health-promoting benefits of low-fat Cheddar cheese. Three batches of cheese were made by employing a non-EPS–producing cheese starter (control), in combination with Lb. plantarum JLK0142 as an adjunct and the purified EPS as an ingredient. Lactobacillus plantarum JLK0142 survived well in cheese, with counts of 7.99 log cfu/g after 90 d of ripening. All experimental cheeses (with adjunct culture or EPS ingredient) had higher moisture, proteolysis, and sensory scores, and lower hardness and cohesiveness compared with the control cheese. Water-soluble extracts from the experimental cheeses outperformed that of the control in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl radicals, and inhibiting α-amylase, angiotensin-converting enzyme, and HT-29 tumor cell growth. Therefore, incorporation of the EPS-producing culture of Lb. plantarum JLK0142 is promising for improvement of low-fat cheese quality and bioactivities.     

Probiotic Cheddar Cheese: Influence of Ripening Temperatures on Proteolysis and Sensory Characteristics of Cheddar Cheeses

ABSTRACT:  Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI L26, Lb. acidophilus 4962, or Lb. acidophilus LAFTI L10 were used as an adjunct in the production of Cheddar cheeses, which were ripened at 4 and 8 °C for 24 wk. Effects of ripening temperatures and probiotic adjuncts on proteolysis and sensory evaluation of the cheeses were examined. Higher ripening temperature increased the level of proteolysis in the cheeses. Product of proteolysis and organic acids released during ripening were shown to be important for the flavor of Cheddar cheeses. There were positive and significant correlations between the levels of soluble nitrogen, lactic, acetic, and butyric acids, percentage hydrolysis of α_s1-CN and β-CN to the scores of cheddary flavor ( P < 0.05). Scores for sour-acid and vinegary flavors were higher in cheeses with the addition of Bifidobacterium sp. or Lb. casei 279 ripened at 8 °C. The scores were positively and significantly correlated to the level of lactic, acetic, and free amino acids in the cheeses ( P < 0.05). The results show that both 4 and 8 °C have potential for use in the ripening of probiotic Cheddar cheeses.     

Survival of Lactobacillus casei,Lactobacillus plantarum and Bifidobacterium bifidum in free and microencapsulated forms on Iranian white cheese produced by ultrafiltration

Survival of probiotic strains Lactobacillus casei ( ATCC 39392 ), Lactobacillus plantarum ( ATCC 8014 ) and Bifidobacterium bifidum ( ATCC 29521 ) was investigated either in microencapsulated or in free form in the Iranian white cheese produced by ultrafiltration technique. The results indicated that the survival of encapsulated probiotic bacteria was higher than free cells. Both free and microencapsulated forms were successful in keeping counts of L. casei, L. plantarum and B. bifidum in the cheese high enough for the therapeutic minimum (10⁶–10⁷ cfu/g) after 60 days. Addition of probiotic adjunct also did not alter the chemical composition, but pH was lower in probiotic cheeses.     

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.