Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.     

The effect of pH,sodium chloride,sucrose, sorbate and benzoate on the growth of food spoilage yeasts

The effects of pH, concentration of NaCl, concentration of sucrose and concentrations of sorbic and benzoic acids on growth were examined for 30 strains of food spoilage yeasts, representingDebaryomyces hansenii, Yarrowia lipolytica, Kloeckera apiculata, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia anomalaandSaccharomyces cerevisiae.Zygosaccharomyces bailiidid not grow at pH 7.0 andZ. rouxii, Kl. apiculataandP. membranaefaciensdid not grow at pH 8.0. OnlyKl. apiculatagrew at pH 1.5–2.0. The remaining species grew at pH 2.5–8.0. None of the species grew at 20% NaCl but strains ofD. hansenii, Z. rouxiiandP. anomalagrew at 15% NaCl.S. cerevisiaeandK. marxianuswere the least salt tolerant, showing no growth at 7.5% NaCl and 10% NaCl, respectively. Medium pH influenced growth in the presence of NaCl. ForY. lipolytica, D. hanseniiandS. cerevisiae, greatest tolerance of NaCl occurred at pH 5.0–7.0, but forKl.apiculata, D. membranaefaciensandZ. bailiibest tolerance occurred at pH 3.0.K. apiculatagrew in the presence of 12.5% NaCl at pH 2.0. All yeasts grew at 50% sucrose, withZ. rouxii, Z. bailiiandP. anomalaandD. hanseniigrowing at 60–70% sucrose. Medium pH in the range 2.0–7.0 had little effect on ability to grow in the presence of high sucrose concentrations.Z. bailiiandY. lipolyticawere the species most tolerant of sorbate and benzoate preservatives (750–1200 mg l⁻¹) at pH 5.0.     

<Emphasis Type="Italic">α</Emphasis>-Galactosidases production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-1

Extracellular and intracellular α-galactosidases were produced by yeast Debaryomyces hansenii UFV-1 grown on different media with several carbon sources. D. hansenii grown in YP-medium (1% yeast extract and 2% peptone) presented maximum cell mass (8.45 mg/mL) after 36 h of cultivation, with lactose as carbon source, followed by sucrose, glucose, raffinose, and galactose. Higher extracellular and intracellular α-galactosidases activities were observed at 48 h of D. hansenii cultivation in YPmedium containing galactose (0.97 and 5.27 U/mL) and lactose (1.28 and 4.88 U/mL), supporting the evidence for the model of induction for the yeast GAL/MEL regulon, such as described in Sacharomyces cereviseae.     

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.     

Lactose hydrolyzing enzymes in Lactobacillus acidophilus strains

Five Lactobacillus acidophilus strains were examined for lactose hydrolyzing enzymes under several growth conditions. All have both β-galactosidase (β-gal) and phospho-β-galactosidase (P-β-gal) activities. Strains W, ATCC 4356 and NCDO 1748 had different amounts of β-gal activity, but all had 100-fold or more higher than P-β-gal activity. In contrast, in strains ATCC 4962 and ATCC 19992, activities of both enzymes were similar. The β-gal synthesis in strains W, 4356 and 1748 was induced by lactose and galactose, while in strains 4962 and 19992 it appeared not to be inducible. At 45°C in the presence of lactose synthesis of only β-gal occurred in strains 4356, 1748 and W. These differences in lactose hydrolyzing enzymes between the strains could be used, along with other characteristics, for taxonomic differentiation between L. acidophilus strains. Studies with L. acidophilus W indicated that cells have maximum β-gal activity at their early stationary phase of growth and when grown in the presence of an inducer and in the absence of oxgall. Selection of strains, type of carbohydrate in growth medium, growth temperature, culture age, and the absence of oxgall during cell growth should be considered in producing L. acidophilus cultures containing high β-gal activity for use as a dietary adjunct by lactose-intolerant individuals and in the manufacture of acidophilus-fermented dairy products.     

The combined effects of pH,NaCl and temperature on growth of cheese ripening cultures of Debaryomyces hansenii and coryneform bacteria

Growth of five strains each of Debaryomyces hansenii, Brevibacterium linens and Corynerbacterium spp. in a laboratory substrate was investigated at combinations of pH, NaCl concentration and temperature similar to those in surface ripening of cheeses. For D. hansenii the effect of pH on growth was found to be insignificant, but a combination of NaCl and temperature had a significant effect. For strains of B. linens and the Corynebacterium spp., the combined effects of pH, NaCl and temperature were found to be significant. All strains of B. linens, C. ammoniagenes and C. flavescens were stimulated by 4.0% (w/v) NaCl. Quadratic polynomial models were developed that described the growth of D. hansenii as a function of NaCl and temperature and growth of B. linens and Corynebacterium spp. as a function of pH, NaCl and temperature. Model validation in the laboratory substrate showed a good agreement between predicted and observed values.     

Development of predictive models evaluating the spoilage-delaying effect of a bioprotective culture on different yeast species in yogurt

Yeast spoilage of fermented dairy products causes challenges for the dairy industry, including economic losses due to wasted product. Food cultures with bioprotective effects are becoming more widely used to help ensure product quality throughout product shelf life. To assist the dairy industry when evaluating product quality throughout shelf life and the effect of bioprotective cultures, we aimed to build stochastic models that provide reliable predictions of yeast spoilage in yogurt with and without bioprotective culture. Growth characterizations of Debaryomyces hansenii, Yarrowia lipolytica, Saccharomyces cerevisiae, and Kluyveromyces marxianus at storage temperatures of 7, 12, and 16°C during a 30-d storage period were conducted in yogurt with and without a bioprotective culture containing Lacticaseibacillus rhamnosus strains. The kinetic growth parameters were calculated using the Buchanan growth model, and these parameters were used as baseline values in Monte Carlo models to translate the yeast growth into spoilage levels. The models were developed using 100,000 simulations and they predicted yeast spoilage levels in yogurt by the 4 yeast types. Each modeled yogurt batch was set to be contaminated with yeast at a concentration drawn from a normal distribution with a mean of 1 log₁₀ cfu/mL and standard deviation of 1 log₁₀ cfu/mL and stored for 30 d at a temperature drawn from a normal distribution with a mean of 6.1°C and a standard deviation of 2.8°C. Considering a spoilage level of 5 log₁₀ cfu/mL, the predicted number of spoiled samples was reduced 3-fold during the first 10 d and by 2-fold at the end of shelf life when a bioprotective culture was added to the yogurt. The models were evaluated by sensitivity analyses, where the main effect factors were maximum yeast population, storage temperature, and yeast strain. The models were validated by comparing the model output to actual observed spoilage data from a European dairy using the bioprotective culture. When the model prediction, based on a mixture of the 4 specific yeast strains, was compared with spoilage data from the European dairy, the observed effect of bioprotective cultures was considerably higher than predicted, potentially influenced by the presence of contaminating strains more sensitive to a bioprotective culture than those characterized here. The developed Monte Carlo models can predict yeast spoilage levels in yogurt at specific production settings and how this may be affected by various parameters and addition of bioprotective cultures.     

Biodiversity of Yeast Mycobiota in “Sucuk,” a Traditional Turkish Fermented Dry Sausage: Phenotypic and Genotypic Identification,Functional and Technological Properties

In this study, yeasts from Turkish fermented sucuks were identified and their functional and technological properties were evaluated. Two hundred fifty‐five yeast isolates were obtained from 35 different sucuk samples from different regions of Turkey. The yeast isolates were determined as genotypic using 2 different polymerase chain reaction (PCR) methods (rep‐PCR and RAPD‐PCR). Functional and technological properties of including proteolytic, lipolytic, and catalase activities, tolerance to NaCl and bile, as well as growing rates at different temperature and pH conditions selected yeast strains were also evaluated. Candida zeylanoides and Debaryomyces hansenii were dominant strains in sucuk samples. All C. zeylanoides and D. hansenii tested could grow at the condition of 15% NaCl and 0.3% bile salt. However, none of the strains were able to grow at 37 °C, even though catalase activity, weak proteolytic and lipolytic activities was still observed. D. hansenii were able to grow only at pH 3, while some of C. zeylanoides could grow at lower pH levels (pH 2). Three and 4 strains of C. zeylanoides showed β‐hemolysis activity and nitrate reduction ability to nitrite, respectively. D. hansenii did not have properties, which are β‐hemolysis, nitrate reduction, or hydrogen sulfide production. Overall, diverse yeast mycobiota present in Turkish fermented sucuk and their functional and technological properties were revealed with this study.     

Influence of substrate and low temperature on growth and survival of verotoxigenicEscherichia coli

Growth and survival of verotoxigenicEscherichia coli(VTEC) were studied in tryptic soy broth (TSB), brain–heart infusion (BHI), and whole milk at 12, 9.5, 8.5, 7.5, 6.5, and 5.5°C (±0.2°C). Colony forming units (cfu) were enumerated by surface plating on eosin methylene blue (EMB) agar plates and incubating at 37°C for 24 h. Growth curves were plotted and lag and generation times were determined. Results indicated that the average generation times for each strain was similar in each medium (4.6±0.9 h) at 12°C. However, at 9.5°C the average generation time differed significantly among the media (P<0.05). The average generation time was longer in BHI compared to TSB and whole milk at 9.5°C (14 h in BHI vs 11 h in milk and TSB). Also, the generation time of individual strains in BHI varied from 9.5–22 h at 9.5°C. Decrease of temperature to 8.5°C resulted in lack of growth of two strains in BHI. At 7.5°C none of the strains grew in BHI, one grew in TSB, whereas all grew in milk. A further decrease of temperature to 6.5°C did not allow growth of any of the strains in TSB or BHI whereas all grew in milk. Addition of 5% lactose to TSB and BHI enabled survival and growth of all strains at 6.5°C. The results of this study demonstrate the influence of growth medium composition on the minimum growth temperature of verotoxigenicE. coli.     

Evaluation and selection of yeasts isolated from dry-cured Iberian ham by their volatile compound production

One hundred and seventeen yeast strains isolated from dry-cured Iberian ham from the four different protected designations of origin of Spain were investigated for their volatile compound production. The yeast strains were grouped into the two main yeast species usually found in this product (Debaryomyces hansenii and Candida zeylanoides) and 10 different biotypes by restriction mitochondrial DNA analysis. Yeast strains were grown in a designed model culture medium under conditions representative of dry-cured ham processing. Volatile compounds were extracted from this medium using solid-phase micro-extraction and were analysed by gas chromatography/mass spectrometry. Marked differences in volatile compound production were found between D. hansenii and C. zeylanoides and between the mitochondrial DNA patterns of these species. Two of the mitochondrial DNA patterns of D. hansenii exhibited the highest production of the volatile compounds involved in the dry-cured flavour. Consequently, these patterns of D. hansenii should be proposed as starter cultures for dry-cured ham.     

Growth and survival of Yersinia enterocolitica at acidic pH

The ability of three strains of Yersinia enterocolitica to survive and grow in tryptic soy broth (TSB) at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 was determined. In addition, experiments were done to assess survival of Y. enterocolitica in mayonnaise, spoonable salad dressing, and tartar sauce. Two of the strains were able to grow in TSB at pH 4.5 and above when incubated at 25°C. Cell concentrations of all strains decreased at lower pH values at this temperature. No growth of Yersinia was observed when TSB was incubated at 5°C for any pH tested, although survival was prolonged. Viable cells were not recovered from artificially contaminated tartar sauce (pH 3.2) or spoonable salad dressing (pH 3.4) at any time. Cells survived for at least 48 h in artificially contaminated mayonnaise (pH 3.8) held at 5°C. No viable cells were recovered after 24 h from mayonnaise held at 21°C.     

Isolation of Casein by Ultrafiltration and Cryodestabilization

Casein was isolated from skim milk by ultrafiltration, storage of the retentate at -8°C for l-4 wk, and centrifuging of thawed samples at 5,000 ×g for 10 min. The extent of cryodestabilization of casein was dependent on the extent of ultrafiltration and storage time at -8°C. Ultrafiltration to a 4× or 6× volume concentration (VCR) ratio resulted in greater than 95% recovery of casein. Casein thus precipitated could be washed with water at 0°C without any significant loss of casein. Removal of lactose caused accelerated casein cryodestabilization in ultrafiltered samples as compared to control samples. Casein precipitated by this method is dispersible in water, whereas acid precipitated casein is not.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

The population change of yeasts in commercial salami

The survival of yeast during a pilot scale production of commercial salami was investigated. A total of 108 distinctive yeast strains were isolated and identified during the processing of the salami. Initially, the number of yeasts remained below 10³cfu g⁻¹but their numbers increased after the 12^thday of maturation, reaching a maximum of 2·0×10⁵cfu g⁻¹at day 20. During maturation, the pH declined from 5·72 to 4·36, water content from 58% to 43%, while the salt content increased by 1%. The number of lactic acid bacteria remained above 10⁵cfu g⁻¹throughout processing and maturation. Of the 108 yeast strains isolated, 22 strains were identified as members of the species Debaryomyces hansenii, being present in all samples taken. In descending numbers, Rhodotorula mucilaginosa, Yarrowia lipolytica and Cryptococcus albidus, were also frequently isolated during processing and maturation.     

Control of Yersinia enterocolitica in raw pork and pork products by γ-irradiation

γ-Radiation response of Y. enterocolitica 5692 and 152 was studied at 0°C and at −40°C in phosphate buffer (pH 7.00) as well as in 10% raw meat/salami homogenate. The strains investigated did not differ in their response and were found to be sensitive to γ-radiation but exhibited a tailing phenomenon in the survival curve. The D₁₀ in homogenate was 0.25 kGy at 0°C. This response was not affected at −40°C. Storage studies of packs, inoculated artificially with heavy inoculum of Y. enterocolitica (10⁶ cfu/g) showed that while samples of salami and cooked ham could be decontaminated at doses of 4 and 3 kGy respectively; cells could not be eliminated from raw pork meat even at the higher dose of 6 kGy. The role of different treatments given prior to irradiation for revival of Y. enterocolitica after irradiation storage was also studied. The dose of 1 kGy at −40°C was efficient in eradicating low numbers (<10³) of naturally occuring Y. enterocolitica from raw pork meat without any revival during storage at refrigeration temperature.     

A model describing Debaryomyces hansenii growth and substrate consumption during a smear soft cheese deacidification and ripening

A mechanistic model for Debaryomyces hansenii growth and substrate consumption, lactose conversion into lactate by lactic acid bacteria, as well as lactose and lactate transfer from the core toward the rind was established. The model described the first step (14 d) of the ripening of a smear soft cheese and included the effects of temperature and relative humidity of the ripening chamber on the kinetic parameters. Experimental data were collected from experiments carried out in an aseptic pilot scale ripening chamber under 9 different combinations of temperature (8, 12, and 16°C) and relative humidity (85, 93, and 99%) according to a complete experimental design. The model considered the cheese as a system with 2 compartments (rind and core) and included 5 state evolution equations and 16 parameters. The model succeeded in predicting D. hansenii growth and lactose and lactate concentrations during the first step of ripening (curd deacidification) in core and rind. The nonlinear data-fitting method allowed the determination of tight confidence intervals for the model parameters. The residual standard error (RSE) between model predictions and experimental data was close to the experimental standard deviation between repeated experiments.     

Lactobacillus plantarum 70810 from Chinese paocai as a potential source of β-galactosidase for prebiotic galactooligosaccharides synthesis

A novel food-grade strain Lactobacillus plantarum 70810 producing β-galactosidase with high transgalactosylation activity was isolated from Chinese paocai. The galactooligosaccharides (GOS) were synthesized by using this enzyme with a maximum yield of 44.3 % (w/w) from 400 g/L lactose at 45 °C for 10 h. The β-galactosidase from this strain was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. It was a heterodimer arrangement of approximately 105 kDa composed of two subunits of 35 and 72 kDa. The optimal pH of the purified β-galactosidase was 8.0 for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis, and optimal temperature was 60 °C and 55 °C, respectively. Its K _m and V _max values for oNPG and lactose were 0.89 ± 0.05 mM, 194 ± 3.0 μmoL/min/mg protein, and 9.88 ± 0.16 mM, 15.88 ± 0.21 μmoL/min/mg protein, respectively. This enzyme was slightly inhibited by the hydrolysis products, that is, glucose and galactose. Since the β-galactosidase from L. plantarum 70810 exhibited higher transgalactosylation activity, strong affinity for lactose and low end-product inhibition, it was suggested to be a potential candidate for the synthesis of prebiotic GOS.     

Influence of temperature on growth rate and lag phase of fungi isolated from Argentine corn

The influence of temperature on the growth of nine strains of fungi belonging to the genera Eurotium, Aspergillus, Penicillium and Fusarium has been investigated for the temperature range 15–35° C. The lag phase and the growth rate were evaluated by using a laboratory medium. The maximum growth rate for E. repens, A. wentii and P. chrysogenum was observed at about 25° C, for P. citrinum near 30° C and for F. semitectum and F. moniliforme between 20 and 25° C. The growth rate of A. niger, A flavus and A. parasiticus increased with increasing temperatures in the range studied. For all strains studied it appeared that the higher the growth rate the lower the lag phase was.     

Enhancement of microbial oil production by alpha-linolenic acid producing Yarrowia lipolytica strains QU22 and QU137

Microbial oils enriched in essential polyunsaturated fatty acids can be used as nutritional complements. In order to improve the lipid yields and lipid profiles of 2 Yarrowia lipolytica strains, the effects of medium components and culture conditions were investigated using shake flasks. Under optimized conditions (cultivation for 3 days at 25oC and 150 rpm, using peptone as a nitrogen source and glucose as a carbon source), the biomass, lipid yield, and lipid content reached 6.75 g/L, 3.48 g/L, and 51.55% for Y. lipolytica QU22, and 6.85 g/L, 3.52 g/L, and 51.38% for Y. lipolytica QU137, respectively. Using a shaking speed of 150 rpm, unsaturated fatty acids constituted 87.96% (QU22) and 87.08% (QU137) of the total lipids produced by these strains, with 9.52 and 7.86% of alpha-linolenic acid, respectively. The strains are suitable candidates for fermentation processes involving essential polyunsaturated fatty acid (PUFA) production.     

Bovine milk procathepsin D: Presence and activity in heated milk and in extracts of rennet-free UF-Feta cheese

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α_s1-I and para-κ-caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.