Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified refrigerated milk

 The growth and activity of two Lactococcus strains and one Leuconostoc strain in CO₂-acidified and non-acidified refrigerated milk were evaluated separately and as a mixed culture to determine their suitability for use as a starter in the manufacture of Afuega’l Pitu, an acid-coagulated Spanish cheese, from refrigerated CO₂-acidified milk. The growth of the strains studied and their production of organic acids were similar in CO₂-acidified and non-acidified refrigerated or fresh milk, indicating that CO₂ treatment does not affect the metabolic activity of the strains. However, refrigeration enhanced the production of acetaldehyde, ethanol and diacetyl in CO₂-acidified and non-acidified milk. The level of diacetyl was also greater in refrigerated CO₂-acidified milk than in refrigerated non-acidified milk. It was concluded that refrigerated milk acidified with CO₂ can be satisfactorily used in the manufacture of Afuega’l Pitu cheese, and that this technique can be also used in the production of other acid-coagulated cheeses. Received: 1 July 1997 / Revised version: 1 September 1997     

Afuega’l Pitu Cheese Quality: Carbon Dioxide Addition to Refrigerated Milk in Acid-coagulated Cheesemaking

The suitability of milk preserved by refrigeration and CO₂ addition for the manufacture of Afuega’l Pitu, a Spanish acid-coagulated short-ripened cheese, was evaluated at pilot scale using an autochthonous cheese starter. Cheeses manufactured, after milk pasteurisation, from vacuum degasified refrigerated CO₂-treated samples (pH 6.2) were compared with two different control cheeses made from pasteurised milk either fresh or refrigerated. The multiplication and acidification capacity of lactic acid bacteria as well as the production of volatile compounds during cheesemaking and ripening were not affected by the previous refrigeration and CO₂-treatment of raw milk nor by the residual CO₂ still present in pasteurised milk after degasification and pasteurisation. Residual CO₂ retarded the proteolysis although no differences on proteolysis and sensory properties were detected at the optimum time of consumption (15 days) between cheeses made from CO₂-treated milk and those made from untreated control milk. CO₂-treatment effectively prevents the decrease in cheese yield caused by the microorganisms present in raw milk of poor microbial quality. These results support the interest of the CO₂-addition in preservation of milk for cheesemaking, specially when it has a high initial microbial load; the method can be satisfactorily used in the manufacture of Afuega’l Pitu cheese and may be extended to the production of other acid-coagulated cheeses.     

Quality of plain yoghurt made from refrigerated and CO2-treated milk

The suitability of milk preserved by refrigeration and CO₂ addition for the manufacture of plain yoghurt was evaluated using two commercial strains of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. Yoghurts manufactured, after milk pasteurisation, from refrigerated CO₂-treated samples (pH 6.15) were compared with two different controls made from pasteurised milk, either fresh or refrigerated. The multiplication and acidification capacity of the starter as well as the evolution of organic acids were not affected by the previous refrigeration and CO₂-treatment of raw milk nor by the residual CO₂ present in pasteurised milk. However, refrigeration enhanced the production of ethanol and diacetyl. No differences on sensory properties were detected through the cold storage between yoghurts made from CO₂-treated milk and those made from refrigerated control milk. It was concluded that refrigerated milk acidified with CO₂ could be satisfactorily used in the manufacture of yoghurt.     

Manufacture of cheese made from CO2-treated milk

Cheeses were manufactured from pasteurised milk (control), pasteurised milk acidified to pH 6.0 with CO₂, and milk acidified to pH 6.0 with CO₂ prior to pasteurisation. Production of cheese from CO₂-treated milk at pH 6.0 reduced the amount of rennet necessary for coagulation by about 75%. Although acidification reduces the amount of lactic acid produced by starter during incubation of milk, no significant differences in lactic acid content were detected between cheeses manufactured from non-acidified or CO₂-acidified milks. Cheeses produced from CO₂-treated milk showed less proteolysis than control cheeses, but no significant differences in sensory characteristics between cheeses were detected.     

Comparative performance of a mixed strain starter in cow's milk, ewe's milk and mixtures of these milks

Lactococcus lactis ssp. lactis IPLA 947, L. lactis ssp. lactis biovar. diacetylactis IPLA 838 and Leuconostoc citreum IPLA 616 and designed for Afuega'l Pitu cheese manufacture from pasteurized milk was assayed in cow's and ewe's milk, and in mixtures containing 10% and 20% ewe's milk in cow's milk. The evolution of microbial counts, pH, titratable acidity, organic acids and volatile compounds production throughout the incubation period was determined. The use of ewe's milk as a culture medium increased the metabolic activity of the starter culture reflected in a higher lactose consumption, significantly higher acidity and some carbon source-derived organic acids and volatile compounds production, as well as in slightly higher starter strains growth, although the latter was not statistically significant. Thus, it is suggested that ewe's milk or mixtures of ewe's with cow's milk can be satisfactorily used in the manufacture of this traditional cheese. Received: 27 August 1999     

Manufacture of cheese made from CO2-treated milk

Cheeses were manufactured from pasteurised milk (control), pasteurised milk acidified to pH 6.0 with CO₂, and milk acidified to pH 6.0 with CO₂ prior to pasteurisation. Production of cheese from CO₂-treated milk at pH 6.0 reduced the amount of rennet necessary for coagulation by about 75%. Although acidification reduces the amount of lactic acid produced by starter during incubation of milk, no significant differences in lactic acid content were detected between cheeses manufactured from non-acidified or CO₂-acidified milks. Cheeses produced from CO₂-treated milk showed less proteolysis than control cheeses, but no significant differences in sensory characteristics between cheeses were detected.

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Herstellung von Käse aus CO₂-behandelter Milch

Zusammenfassung Käse wurde in drei verschiedenen Varianten hergestellt: aus pasteurisierter Milch (Kontrollkäse) sowie aus mit CO₂ auf pH 6.0 angesäuerter Milch, die nach der Säuerung pasteurisiert wurde bzw. angesäuerter Milch, die im voraus pasteurisiert worden war. Es wurde beobachtet, daß die für die Koagulation erforderliche Labmenge bei der Herstellung von Käse aus durch CO₂ auf pH 6.0 angesäuerter Milch, im Vergleich mit Kontrollkäse, auf 75% abnimmt. Der Gehalt der Milch an Starter-Kulturen nahm durch die CO₂-Behandlung ab. Allerdings konnten keine signifikanten Unterschiede im Milchsäuregehalt zwischen den aus nicht angesäuerter bzw. angesäuerter Milch hergestellten Käsen festgestellt werden. Aus CO₂-behandelter Milch hergestellter Käse zeigte geringere Proteolyse als der Kontrollkäse, jedoch konnten keine signifikanten sensorischen Unterschiede zwischen den untersuchten Varianten festgestellt werden.

     

Changes induced by high hydrostatic pressure in acidified and non-acidified milk during Oaxaca cheese production

Oaxaca cheese, produced using the pasta filata method, is a very popular Mexican dairy products. In this work, the effect of high hydrostatic pressure (HHP) and the acidification process before and after HHP treatment of raw cow milk was studied at different pressure levels (150, 300 and 500 MPa) and holding times (10 and 30 min). Clotting time, proximal composition, microstructure, secondary protein structure and electrophoretic profile were evaluated. HHP did not influence clotting time in samples acidified before HHP, but it appears to have a positive effect at lower pressure treatments on non-acidified milk. Moisture, protein and fat were similar in cheeses treated at most HHP conditions regardless of the acidification. HHP did not influence the microstructure of cheese and the secondary structure of proteins. The use of HHP during the manufacture of Oaxaca cheese allowed preserving quality parameters evaluated without advantages in processing time and the product's proximal composition.     

Characteristics of carbonated fermented milk and survival of probiotic bacteria

The carbonation of pasteurised milk was evaluated as a method for improving bacterial viability in fermented milk added with probiotic bacteria (Lactobacillus acidophilus and/or Bifidobacterium bifidum). The behaviour of microorganisms during fermentation and cold storage, and the biochemical and sensory properties of the products were assessed. In AT (Streptococcus thermophilus/L. acidophilus) and ABT (S. thermophilus/L. acidophilus/B. bifidum) products, the fermentation times to decrease the pH to 5 were significantly lowered when CO₂ or lactic acid was added to milk. The higher acidity levels of carbonated (as a result of production of carbonic acid) and lactic acidified samples enhanced growth and metabolic activity of the starter during fermentation and was the reason for this reduction in incubation time. Cell counts of S. thermophilus, L. acidophilus and B. bifidum gradually decreased through the cold storage of carbonated and non-acidified fermented milk, although the counts were always higher than 10⁶ viable cells g⁻¹. The CO₂ did not exert any influence on the viability of S. thermophilus and L. acidophilus in AT fermented milk stored at 4°C but the presence of B. bifidum and CO₂ in ABT-type products was associated with lower viability of L. acidophilus during the refrigerated storage. The higher acetate concentrations of ABT products made with non-acidified milk as compared with the carbonated products could have contributed to major survival of L. acidophilus in the former. The use of milk acidified with CO₂ had no detrimental effects on the sensory properties of ABT fermented milk. Therefore, we concluded that the carbonation of pasteurised milk prior to the starter addition could be satisfactorily used to reduce the manufacture time of fermented milk.     

Seasonal changes in the fat composition of Lacha sheep's milk used for Idiazabal cheese manufacture

 The effect of the seasonality of lacha sheep's milk production on the milk-fat composition was studied. This raw milk is used to manufacture Idiazabal cheese, a typical product of the Basque Country of northern Spain. Milk samples were taken, prior to cheesemaking, from a refrigerated tank of mixed milk from different flocks in a cheese factory in February, April and June. The triglyceride and fatty acid composition of the milk were analysed. The results showed that the molecular characteristics of the milk-fat components, the degree of unsaturation and the chain length changed with the time of the year. The June milk had a higher content of unsaturated and long-chain fatty acids and triglycerides. The April and February milks were very similar, with a higher content of saturated and short-chain fatty acids and triglycerides. The differences in animal feeding are probably the most important factor affecting milk composition because in June flocks are fed mainly in natural pastures while in April and February the ewes are also fed additional commercial feeds. Idiazabal cheeses were considerably different from February to June, suggesting a relationship between the free fatty acid composition of the cheese, its sensorial properties and the fat composition of the raw milk used in cheesemaking. Received: 18 June 1999     

Influence of cryoprotectants on the viability and acidifying activity of frozen and freeze-dried cells of the novel starter strain Lactococcus lactis ssp. lactis CECT 5180

  Lactococcus lactis ssp. lactis CECT 5180 is a novel starter strain with highly desirable technological properties, selected to be used a starter culture for the manufacture of Afuega'l Pitu cheese, a farmhouse cheese very popular in Asturias (Northern Spain). In order to optimise biomass preservation of this strain the effect of different substances on the viability and acidifying activity of preserved cells (by freezing or freeze-drying) was evaluated. On the whole, supplementing reconstituted skimmed milk with certain substances enhances its own highly-protective effect, to different degrees depending on the compound used. In fact, substances as β-alanine for freeze-drying procedures and sugars, glutamic acid or meat extract (only at –80  °C) for freezing purposes, showed a highly suitability for preservation processes, not only as high-survival providers but also in relation to the reduction in the lag phase of cultures in milk. Received: 17 January 2000     

Rational selection of Leuconostoc strains for mixed starters based on the physiological biodiversity found in raw milk fermentations

The technological abilities of eleven wild Leuconostoc strains isolated from artisanal Afuega'l Pitu cheese, a homemade acid-coagulated cheese from raw milk from Asturias (northern Spain), were studied in order to test their potential application as components of dairy starters. Metabolic activity, production of flavour compounds, resistance to NaCl, acid, nisin and freezing, as well as genetic biodiversity were investigated. Marked differences among all tested strains reflected the existing biodiversity in naturally fermented products. After evaluation of their performance, strains IPLA567 and IPLA979 revealed the best properties to be used use in mixed dairy starter cultures. These two strains were able to ferment lactose and galactose and produced larger amounts of lactic acid than the others. Moreover, they showed high tolerance levels to NaCl, acid and nisin, although their resistance to freezing was slightly lower than the other strains. This study evidences the fact that natural environments can be considered as a proper source of new strains, which may help the dairy industry to widen their fermented products range.     

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style)

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log₁₀ cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log₁₀ cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log₁₀ cfu/g after whey drainage and during ripening and storage decreased by 2.9 log₁₀ cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.     

Dissimilation of organic acids by dairy lactic acid bacteria

One hundred and eleven different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus and Lactobacillus, were examined for their ability to degrade 10 organic acids by detecting CO₂ production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxoglutarate, or mucate. Malate, fumarate, citrate, gluconate, tartrate and pyruvate were variably attacked. A malo-lactic fermentation was brought about by two thirds of S. cremoris and S. lactis strains as well as L. casei but not by L. buchneri or L. brevis. One third of the lactic streptococci examined, i.e. S. cremoris AM₂, ML₈ and SK₁₁ were capable of diacetyl production. They required glucose for cleavage of citrate, whereas Leuconostoc citrovorum and S.faecalis did not. S. cremoris differed from S. lactis in not producing CO₂ from gluconate. From all the lactic acid bacteria examined, only L. plantarum dissimilated tartrate. Production of acetoin and diacetyl is a more reliable evidence for assessing the degradation of pyruvate, compared to detection of evolved CO₂. Facultatively heterofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas the obligately heterofermentative lactobacilli did not, a character that would be of taxonomic value. Glucose fermented by both the facultatively and obligately heterofermentative lactobacilli did not prove to be a metabolic source of acetoin and diacetyl. The facultative heterofermenters degraded pyruvate in the presence of glucose with lactate as the major product together with a mean of acetate of 4.1%, ethanol of 7.9%, acetoin of 1.7% and diacetyl of 2.6% yield on a molar basis after 60 days at 30°C. L. brevis produced acetate and lactate. The role of S. cremoris and the facultatively and obligately heterofermentative lactobacilli in flavour development during the ripening of many cheese varieties has been confirmed.     

An indirect competitive ELISA for the detection of cows’ milk and caseinate in goats’ and ewes’ milk and cheese using polyclonal antibodies against bovine γ-caseins

 An indirect competitive enzyme-linked immunosorbent assay (ELISA) method was developed for the detection of bovine milk and caseinate in goats’ and ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits and chickens against bovine γ₃-casein. In a first affinity chromatography step, antibodies recognizing caseins were absorbed on bovine casein-Sepharose. From the dialysed eluate, antibodies crossreacting with ewes’ and goats’ milk protein were completely removed by immunoadsorption onto stationary phases containing ovine casein and protein extracted from genuine ewes’ and goats’ milk cheese. The detection limit of the ELISA test was 0.1% and the method was applied successfully during an EU collaborative study of the evaluation of methods for the detection of cows’ milk. Received: 15 February 1996     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

Preservation of raw milk with CO2

The effect of CO₂ on the growth of psychrotrophic milk spoilage organisms was studied, both in raw fresh milk and in pure cultures of three species ofPseudomonas growing in sterilised milk. Changes of sensory properties of CO₂-treated samples after heat treatment were also analysed. Inhibition of psychrotrophic growth at 7 °C in milk treated with CO₂ to a pH 6.2 or 6.0 was impaired by a gradual reduction of the CO₂ content during storage. Growth inhibition was considerably improved by pH adjustment at 24-h intervals. Sensory analysis showed significant differences between non-acidified and acidified samples after heat treatment at 75 °C for 20 s or 110 °C for 5 min. No sensory differences were found between non-acidified and acidified milks degassed before heat treatment.     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

Effect of the use of carbon dioxide on Prato cheese making

The effects of the addition of carbon dioxide (CO₂) under pressure (1.6 × 10⁵ Pa at 8 °C) to pasteurised Prato cheese milk (pH 6.0) was investigated through 120 d of refrigerated storage. The addition of CO₂ decreased the curd formation time (30 min), the total manufacturing time (47 min), and the pH of Prato cheese, thus leading to reduced moisture content. The CO₂ treated cheese showed higher firmness and fracturability due to the greater whey loss. In contrast, the microorganism counts, cheese yield, protein loss, cohesiveness, springiness, and gumminess were not significantly affected by the treatment. For the lactose fermentation, no significant differences were observed. The addition of CO₂ did not change the proteolysis indexes, and no significant differences were observed in the sensory acceptance of the CO₂ treated cheese, which was well accepted by consumers.     

Lactic acid production and rheological properties of yogurt made from milk acidified with carbon dioxide

The influence of milk acidification up to pH 6.0 with CO₂ on D ‐ and L ‐lactic acid production and lactose consumption by yogurt starter, changes in the pH, and rheological and sensory properties of yogurt were studied. A slight influence of CO₂ on lactic acid production during yogurt manufacture was detected. No significant changes in lactic acid concentration were observed during storage, although the final concentration was significantly lower in control than in pH 6.2 and 6.0 acidified samples. A great influence of CO₂ on D ‐lactic acid production was not observed. Yogurt manufactured from milk with lower pH values showed lower final pH values after 7 days of storage. The viscosity was similar in all analysed samples. No significant differences in sensory characteristics between unacidified and acidified yogurts were detected. © 1999 Society of Chemical Industry