Phytochemical and Antimicrobial Evaluation of Aqueous and Organic Extracts of Calotropis procera Ait Leaf and Latex

The aqueous and ethanol extract of Calotropis procera leaf and latex were investigated. The leaves and latex from the plant were tested for antimicrobial activities. The bioactive constituents extracted from the leaf and latex were tested against pathogenic organisms (Eschericia coli, Salmonella typhi, Bacillius subtilis, Candida albicans, Aspergillus niger) using the Agar well diffusion method. The ethanolic latex extract showed significant activity against all the test organisms. The results revealed that ethanol is a more effective extractive solvent for antimicrobial activity of leaf and latex of C. procera. The ethanol extract of the latex gave the widest zone of inhibition (21 mm) against B. subtilis. All the extracts inhibit the growth of all the organisms except B. subtilis of which the aqueous extract has no effect. The Minimum Inhibitory Concentration (MIC) for the latex extract was between 3 and 7.5 mg/ml for bacteria, and 5.0 to 7.0 mg/ ml for fungi. For the leaf extract the MIC for bacteria was between 5.0 and 10.5 mg/ml and 11 and 15 mg/ml for fungi. The results also showed an increase in antimicrobial activity with increase in temperature. This study therefore revealed that C. procera latex extract demonstrated strong and better inhibitory activity on the test organisms than the leaf extract. These findings therefore provide an explanation for the traditional medicinal use of C. procera extracts.     

Applicability of extracts from Centaurea calcitrapa in ripening of bovine cheese

Aqueous extracts obtained from cell suspension cultures of Centaurea calcitrapa were used as proteolytic additive in the manufacture of a commercial bovine cheese, coagulated with animal rennet and typically ripened for 28 d. The cheese was assessed in comparison to standard cheese for two levels of addition of said extract, viz. 0.61 and 1.22 mg of total protein mL⁻¹. The qualitative and quantitative evolutions of the nitrogen fractions were monitored in the experimental cheeses throughout the whole ripening period. In general, the chemical compositions of the cheeses were different depending on the amount of extract used, but no significant differences could be detected in the ripening index. With regard to electrophoretic profiles, the two types of cheese could be distinguished until up to ca. 7 d of ripening, but differences did essentially vanish by 28 d.     

Survival of Listeria monocytogenes in Galotyri,a traditional Greek soft acid-curd cheese,stored aerobically at 4°C and 12°C

Galotyri is a traditional Greek soft acid-curd cheese, which is made from ewes’ or goats’ milk and is consumed fresh. Because cheese processing may allow Listeria monocytogenes post-process contamination, this study evaluated survival of the pathogen in fresh cheese during storage. Portions (0.5 kg) of two commercial types (<2% salt) of Galotyri, one artisan (pH 4.0±0.1) and the other industrial (pH 3.8±0.1), were inoculated with ca. 3 or 7 log cfu g⁻¹ of a five-strain cocktail of L. monocytogenes and stored aerobically at 4°C and 12°C. After 3 days, average declines of pathogen's populations (PALCAM agar) were 1.3–1.6 and 3.7–4.6 log cfu g⁻¹ in cheese samples for the low and high inocula, respectively. These declines were independent (P>0.05) of the cheese type or the storage temperature. From day 3, however, declines shifted to small or minimal to result in 1.4–1.8 log cfu g⁻¹ of survivors at 28 days of storage of all cheeses at 4°C, indicating a strong “tailing” independent of initial level of contamination. Low (1.2–1.7 log cfu g⁻¹) survival of L. monocytogenes also occurred in cheeses at 12°C for 14 days, which were prone to surface yeast spoilage. When ca. 3 log cfu g⁻¹ of L. monocytogenes were inoculated in laboratory scale prepared Galotyri of pH ≅4.4 and ≅3% salt, the pathogen died off at 14 and 21 days at 12°C and 4°C, respectively, in artisan type cheeses fermented with the natural starter. In contrast, the pathogen survived for 28 days in cheeses fermented with the industrial starter. These results indicate that L. monocytogenes cannot grow but may survive during retail storage of Galotyri despite its low pH of or slightly below 4.0. Although contamination of Galotyri with L. monocytogenes may be expected low (<100 cfu g⁻¹) in practice, that long-term survival of the pathogen in commercial cheeses was shown to be unaffected by the artificial contamination level (3 or 7 logs) and the storage temperature (4°C or 12°C), which should be a concern.     

A comparison of the chemical,microbiological and sensory characteristics of bovine and ovine Halloumi cheese

Commercial samples of fresh and mature Halloumi cheeses made from ovine or bovine milk were studied in order to establish their chemical, microbiological and sensory characteristics. Significant differences were observed between the two types of Halloumi cheese both when fresh and mature. The free volatile fatty acid (FVFA) content of the cheeses increased with maturation from 483 to 1356 mg kg⁻¹ for the ovine product, but lower values (380–1248 mg kg⁻¹) were found in the bovine cheese. During maturation for 40 days, Enterococcus faecium, which dominated the microflora of fresh ovine cheese, was replaced by lactobacilli, including a new species, Lactobacillus cypricasei, which was not found in the bovine samples. Fewer than 100 cfu g⁻¹ lactic acid bacteria (LAB) were present in the fresh bovine cheeses, but a microflora dominated by lactobacilli developed with time. Yeast counts in the mature ovine and bovine cheeses reached 2.3–2.8×10⁵ cfu g⁻¹ and, as some of the yeasts were proteolytic and/or lipolytic, it was assumed that they were having a positive impact of the flavour of the cheeses. The sensory panel distinguished significant differences in texture and flavour between the fresh and mature samples of both ovine and bovine cheeses and, overall, there was a significant preference for the ovine brand.     

Lamb rennet paste in ovine cheese manufacture. Lipolysis and flavour

In a preliminary study with commercial ewe's milk cheeses, there were statistically significant differences in the sensory evaluation scores and the amounts of short-chain (C4–C10) free fatty acids (FFAs) between cheeses made with lamb rennet paste or bovine rennet. Experimental ewe's milk cheeses were manufactured with 2 levels of artisanally produced lamb rennet paste and 2 levels of bovine rennet in 50 L vats, with a manufacturing replicate done within 1 week. Total coagulating activity was 2500 RU for cheeses manufactured with a high amount of rennet or 1000 RU for cheeses manufactured with a low amount of rennet. The two batches of lamb rennet pastes used had 4.0 (early Spring) and 6.5 U g⁻¹ lipase (late Spring), whereas no lipase activity was detected in bovine rennets. The total concentration of FFAs in cheeses manufactured with lamb rennet paste was significantly higher than in cheeses manufactured with bovine rennet, regardless of ripening time and time of the year. Lipolysis in the former cheeses increased with the total units of lipolytic activity added. The increase in lipolysis in cheeses made with lamb rennet was primarily due to higher amounts of short-chain fatty acids (C4–C10). Butyric acid was the main FFA in cheeses made with lamb rennet paste, representing 34.5 μmol 100 μmol⁻¹ (low level of rennet) and 44.2 μmol 100 μmol⁻¹ (high level of rennet) of the total FFAs, whereas it represented only 17 μmol 100 μmol⁻¹ of the total FFAs in all cheeses made with bovine rennet. The concentration of individual FFAs of chain length <C12 were significantly higher in cheeses made with lamb rennet paste than in cheeses made with bovine rennet. Cheeses made with lamb rennet paste received significantly higher intensity scores for odour and flavour intensity, sharp odour, ‘piquant’ and bitter flavours and ‘natural rennet’ odour and flavour.     

Effect of cook temperature on primary proteolysis and predicted residual chymosin activity of a semi-hard cheese manufactured using thermophilic cultures

Novel semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus as starter cultures and with cook temperatures of 47, 50 or 53 °C. There was a progressive and significant degradation of both α_s1- and β-caseins during ripening of all cheeses. Increasing cook temperature significantly reduced degradation of α_s1-casein during ripening, in the order 53<50<47 °C, as measured by densitometric analysis of urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms. Mean levels of primary proteolysis, as measured by amounts of pH 4.6-soluble N, were also significantly reduced. A mathematical model, incorporating changes in pH and temperature during manufacture of the cheeses, predicted near to total inactivation of residual chymosin as a result of the cooking profiles used in cheesemaking. Increasing cook temperature did not completely inhibit primary proteolysis or hydrolysis of α_s1-casein to α_s1-casein (f24–199) during ripening, although these reactions were slowed.     

Effect of high pressure treatment applied on starter culture or on semi-ripened cheese in the quality and ripening of cheese in brine

Cheese ripening acceleration is of continuous interest for the industry. High-pressure (HP) treatment of starter cultures used in cheese-manufacturing offers the potential to accelerate ripening by increasing the activity of their intracellular peptidases that contribute in the development of desired cheese organoleptic characteristics.The objective of the present research was the investigation of the effect of HP treatment (200 MPa-20 °C - 20 min) directly on white brined cheese or on the starter culture used for its manufacture (Str. thermophilus:L. lactis:L. bugaricus 2:1:1). For this purpose, the microbial, textural, physicochemical and organoleptic characteristics and proteolysis were assessed during the 2nd stage of ripening in cold stores. Control cheese without any treatment was also studied.Cheeses made with HP-treated starters had increased secondary proteolysis. Organoleptic scoring of these cheeses was higher during the whole storage period compared to control and HP-treated cheese. Their superiority was evident even at the early stages of ripening in cold stores, since no bitterness was detected. On the contrary, although HP treated cheeses showed the highest increase in aminopeptidases activities, this was not correlated with the studied ripening indices or the organoleptic characteristics.According to the results, HP-treated starter culture can accelerate proteolysis and potentially the ripening of cheese-in-brine.Industrial relevanceThe data obtained from this work suggest that application of HP treatment under optimized conditions on cheeses in brine starter cultures or on whole cheeses can be effectively used for the production of products with reduced ripening time. This is of great importance for the cheese industries, since the storage period for ripening is long (higher than two months), while applying HP treatment as suggested in this study, this time may be reduced to less than one month, producing cheeses of superior quality.     

Use of high-pressure-treated milk for the production of reduced-fat cheese

Pasteurized (65°C, 30 min), pressurized (400 MPa, 22°C, 15 min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate of protein breakdown than the cheese made from pasteurized milk, that might be mainly attributed to a higher level of residual rennet. Hardness of the experimental cheeses, as determined by both the sensory panel and instrumental analyses, decreased as the moisture content and proteolytic degradation of the cheese increased (pasteurized>pressurized>pasteurized–pressurized). In general terms, pressurization of reduced-fat milk prior to cheese-making improved cheese texture and thus accounted for a higher overall acceptability, except for the cheeses made from pasteurized–pressurized milk at 60 d of ripening, whose acceptability score was adversely affected by bitterness.     

Effect of combinations of high-pressure treatment and bacteriocin-producing lactic acid bacteria on the survival of Listeria monocytogenes in raw milk cheese

The combined effect of high-pressure (HP) treatment and bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Listeria monocytogenes Scott A in cheeses made from raw milk that was inoculated with the pathogen at 4.80 log cfu mL⁻¹, a commercial starter and one of seven strains of BP-LAB was investigated. On day 3, the counts of L. monocytogenes were 7.03 log cfu g⁻¹ in a control cheese (without BP-LAB, not HP treated), 6.06–6.74 log cfu g⁻¹ in cheeses with BP-LAB, 6.13 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 300 MPa, 2.01 log cfu g⁻¹ in a cheese without BP-LAB and treated on day 2 at 500 MPa, 3.83–5.43 log cfu g⁻¹ in cheeses with BP-LAB and treated on day 2 at 300 MPa, and 1.81 log cfu g⁻¹ or less in cheeses with BP-LAB and treated on day 2 at 500 MPa. HP treatment was more effective on day 51 than on day 2.     

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

Effect of cook temperature on starter and non-starter lactic acid bacteria viability,cheese composition and ripening indices of a semi-hard cheese manufactured using thermophilic cultures

Semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus cultures and their ripening was characterised. During cheese manufacture, curds were cooked to a maximum temperature of 47, 50 or 53 °C, pre-pressed under whey at pH 6.15, moulded, pressed and brined. Increased cook temperature resulted in increased manufacture time, a significantly reduced growth rate of S. thermophilus during manufacture in the order 47≈50 °C>53 °C and in significantly lower mean viable cell counts of S. thermophilus up to 56 d of ripening. Increasing cook temperature had no significant effect on mean viable cell numbers of L. helveticus or non-starter lactic acid bacteria (NSLAB). Cheeses produced from curds cooked to 47 °C had significantly higher levels of moisture in non-fat substances (MNFSs), salt-in-moisture and a significantly lower pH and levels of butyrate compared with cheeses produced from curds cooked to 50 or 53 °C.     

An investigation of the plasmin–plasminogen system in caprine milk and cheese

The variability in activities of plasmin (PL) and plasminogen (PG) in bulk milk samples from different breeds of goats was investigated. The mean PL activity was higher (19.56±4.72 U g⁻¹) than in milk from other ruminant species, while PG-derived activity was, surprisingly, lower (12.84±5.31 U g⁻¹). No significant differences in PL and PG levels were observed among goat breeds, but PL activity and PL/PG ratio increased in late lactation compared with early lactation. PL activities in pilot-scale curds (acid or rennet) and in some Italian cheeses made from caprine milk were also measured. Generally, acid curds and cheeses had lower residual PL and PG-derived activities than rennet curds and semi-hard cheeses. In addition, the PL/PG ratio was greatly reduced in rennet curds, due to a higher extent of PG-derived activity. Measurement of proteolysis of β-casein in curds confirmed that more extensive proteolysis occurred in rennet curds, which had higher residual PL activities.     

Study of the chemical composition,proteolysis, lipolysis and volatile compounds profile of commercial Reggianito Argentino cheese: Characterization of Reggianito Argentino cheese

Reggianito is a typical variety of grana-type hard cheese produced in Argentina. It is the most exported and due to its organoleptic characteristics is very appreciated by the consumers. The objective of this study was to characterise the global composition, lipolysis, proteolysis and volatile compound profiles of commercial Reggianito cheeses from different dairy plants.Statistical differences (P ? 0.05) in some physicochemical parameters, nitrogen fractions and FFA levels among commercial brands were detected. The volatile profiles were studied by SPME–GC–MS/FID. A total of 53 compounds were identified, the majority belonging to the groups of ketones, alcohols, acids, esters and aldehydes. All these compounds have been reported in Italian grana-type cheeses. Visualization of the analytical results was performed by principal component analysis. This analysis clustered cheese samples according to dairy plants. This fact could be, among other factors, consequences of differences in technologies and ripening time of different manufacturers.     

Influence of lupin-based milk alternative heat treatment and exopolysaccharide-producing lactic acid bacteria on the physical characteristics of lupin-based yogurt alternatives

In the present study we investigated the influence of heat treatment of lupin-based (LB) milk alternatives and different exopolysaccharide (EPS)-producing lactic acid bacteria on the physical characteristics of set-type LB yogurt alternatives. LB milk alternatives, obtained from protein isolate of Lupinus angustifolius cv. Boregine, were either pasteurized at 80 °C for 60 s or ultra-high temperature (UHT) heated at 140 °C for 10 s and was fermented with Lactobacillus plantarum TMW 1.460 and 1.1468, Pediococcus pentosaceus BGT B34 and Lactobacillus brevis BGT L150. Fermentation duration was strongly affected by heat treatment: different strains needed between 25 to 35 h in UHT LB milk alternative to reach a pH of 4.5 compared to 14 to 24 h in pasteurized LB milk alternative. EPS extraction revealed slightly higher amounts of EPS for UHT LB yogurt alternatives (~ 0.5–0.9 g/l; pasteurized: ~ 0.4–0.7 g/l). The more intensive heat treatment (UHT) resulted also in better rheological (apparent viscosity, hysteresis loop area, flow point, elastic, viscous and complex modulus) and textural properties (firmness, consistency, cohesiveness and index of viscosity) of the investigated LB yogurt alternatives. Furthermore, LB yogurt alternatives out of UHT milk alternative revealed a lower tendency to syneresis, measured with siphon and centrifugation method. This work contributes to the fundamental knowledge of the textural properties of LB yogurt alternatives.     

Evolution of lipolysis during the ripening of traditional Feta cheese

Lipolysis was studied during ripening of traditional Feta cheese produced in two small dairies, A and B. The cheeses were made from a thermized mixture of ewes’/goats’ milk by using yoghurt as starter and artisanal rennet from lambs’ and kids’ abomasa (cheese A) or mixed artisanal rennet with calf rennet (cheese B).The acid degree value and the free fatty acids (FFA) contents in both cheeses increased sharply up to 18 d (pre-ripening period at 15 °C) and continued to increase throughout ripening. In both mature cheeses, acetic acid was found at high levels (13–18% of the total FFAs). However, except for this, all FFA contents differed significantly (P < 0.05) between the two cheeses throughout ripening. The levels of individual and total C2:0–C8:0, C10:0–C14:0 and C16:0–C18:2 fatty acids were significantly higher (P < 0.05) in cheese A than in cheese B. Presumably the difference, especially in the C2:0–C8:0 content, was due mainly to the type of the rennet used. Butyric acid was the dominant FFA in cheese A (20% of the total FFAs at 120 d), while the most abundant FFAs in cheese B were capric (18%) and lauric acid (18%). In general, the lipolysis degree of the two cheeses was higher than those reported for the industrially-made Feta cheese.In organoleptic evaluation, cheese A had a piquant taste that was attributed to its high content of butyric acid and showed a significantly (P < 0.05) higher total score than cheese B.     

A preliminary study on the role of alkaline phosphatase in cheese ripening

A preparation of exogenous alkaline phosphatase (ALP), containing 17,500 mU L⁻¹, was added to pasteurized milk (PM) to study its role in cheese ripening. Three miniature Cheddar-type cheeses were made from PM containing no added ALP (control), PM plus 23 μL ALP (T1), to give ALP concentration similar to that in raw milk, and PM plus 46 μL ALP (T2). Milk, after addition of ALP, was held at 6 °C for 12 h before cheese manufacture and the experiment was replicated three times. The control, T1 and T2 milks contained ALP activity of 415, 2391 and 4705 mU L⁻¹, respectively. The addition of ALP to PM caused significant (P<0.05) changes in moisture content of miniature cheeses but did not cause any changes in protein content. Levels of water-soluble N during ripening of the cheeses were similar for control, T1 and T2 cheeses. The concentration of amino acids was not affected by the level of ALP present in milk. However, reversed-phase HPLC showed differences in the peptide patterns of control, T1 and T2 cheeses, suggesting a role of ALP in cheese ripening. The results suggest that ALP may play a role in cheese ripening, but further studies are needed to confirm this.     

Inhibition of Chymosin and the Coagulation of Para-Casein Micelles by Anions

The effect of monovalent anions on the coagulation of para-casein micelles was determined by monitoring light transmission after adding different Ca salts (40 mM) to partially hydrolyzed casein micelles. The release of macro-peptide by chymosin was quantified using fluorescamine. The average rate of the chymosin-initiated coagulation of casein micelles, approximated by the reciprocal of clotting time, was determined as a function of anion type and concentration to determine the simultaneous effects of anions on chymosin velocity and para-casein micelle aggregability.Chymosin velocity and the coagulation of para-casein micelles were progressively inhibited by anions; the larger the anion, the greater the inhibition (SCN⁻ > NO₃⁻ > Br⁻ > Cl⁻). This is attributed to the binding of anions to cationic binding sites on kappa-casein and para-kappa-casein. The relative effect of the larger anions compared with that of Cl⁻ on the average rate of the chymosin-initiated coagulation of casein micelles increased with anion concentration (6 to 120 mM) but became limited at 120 mM. In the presence of 120 mM SCN⁻, NO₃⁻ and Br⁻, the average rate of coagulation was 26, 59, and 78% of that obtained with 120 mM Cl⁻.The marked sensitivity of the interactions between chymosin and kappa-casein and between para-casein micelles to anion type support conclusions that cationic sites on kappa-casein are important in the mechanism of the chymosin-initiated coagulation of casein micelles. Thus, monovalent anions may be useful to elucidate the mechanism of protein-protein interactions in food systems.     

Activation energy measurements in rheological analysis of cheese

Activation energy of flow (E_a) between 30 and 44 °C was calculated from temperature sweeps of cheeses with contrasting characteristics to determine its usefulness in predicting rheological behavior upon heating. Cheddar, Colby, whole milk Mozzarella, low-moisture part-skim Mozzarella, Parmesan, soft goat, and Queso Fresco cheeses were heated from 22 to 70 °C, and E_a was calculated from the resulting Arrhenius plots. Protein and moisture content were highly correlated with E_a. The E_a values for goat cheese and Queso Fresco, which did not flow when heated, were between 30 and 60 kJ mol^?1. Cheddar, Colby, and the Mozzarellas did flow upon heating, and their E_a values were between 100 and 150 kJ mol^?1. Parmesan, the hardest cheese, flowed rapidly with heat and had an E_a > 180 kJ mol^?1. E_a provides an objective means of quantitating the flow of cheese, and together with elastic modulus and viscous modulus provides a picture of the behavior of cheese as it is heated.     

Controlled permeabilization of Lactococcus lactis cells as a means to study and influence cheese ripening processes

Cells of Lactococcus lactis were permeabilized by perturbing the membrane structure through the delipidating action of n-butanol to an extent that allowed normally excluded peptidase substrates to enter the cells and be accessible to intracellular enzymes. The degree of permeabilization of cells depended on the concentration of the solvent used, the duration of the treatment, the density of the cell suspension, pH and temperature. This was indicated by the optimum or maximum activities of the intracellular peptidases aminopeptidase N and aminopeptidase X. The possible usefulness of permeabilized cells for cheese-ripening studies was demonstrated with cells that were treated with 5 ml L⁻¹ n-butanol at pH 6.5 and 25 °C using a mixture of chymosin-generated primary cheese peptides as substrate. Production of amino acids could be correlated with conversion of peptides that had entered the permeabilized cells and reflected the course of amino acid production in cheese.