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.     

Chemical analysis and sensory evaluation of Cheddar cheese produced with Lactobacillus acidophilus,Lb. casei,Lb. paracasei or Bifidobacterium sp.

The sensory properties of probiotic Cheddar cheeses made using Lactobacillus acidophilus 4962, Lb. casei 279, Bifidobacterium longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 or B. lactis LAFTI^® B94 were assessed after ripening for 9 months at 4 °C. Probiotic cheeses except those with Lb. acidophilus 4962 were significantly different (P<0.05) from the control without any probiotic organism. Acceptability of probiotic cheese with Lb. casei 279 was significantly lower (P<0.05) than that of the control cheese with bitterness and sour-acid taste as the major defects. Concentration of acetic acid in probiotic cheeses was higher (P<0.05) than the control cheese. Vinegary scores did not influence the acceptability of the cheeses (P>0.05). Increased proteolysis in probiotic cheeses did not influence the Cheddary attribute scores (P>0.05). There were positive correlations (P<0.05) between the scores of bitterness and the level of water-soluble nitrogen.     

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

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

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Novel casein-derived peptides with antihypertensive activity

In this study, we report novel casein-derived peptide sequences with angiotensin converting enzyme (ACE)-inhibitory activity and antihypertensive activity demonstrated in spontaneously hypertensive rats (SHR). The peptides were obtained by enzymatic hydrolysis of total isoelectric casein with pepsin. To identify ACE-inhibitory peptides, the casein hydrolysate was fractionated by semi-preparative high performance liquid chromatography, and 44 (CN) peptides contained in the active fractions were sequenced by using an ion trap mass spectrometer. Among the identified peptides, three sequences, that corresponded to α_s1-CN f(90–94) (RYLGY), α_s1-CN f(143–149) (AYFYPEL), and α_S2-CN f(89–95) (YQKFPQY), showed IC₅₀ values as low as 0.71 μm, 6.58 μm, and 20.08 μm, respectively. These three peptides also exerted antihypertensive activity when they were orally administered to SHR at a dose of 5 mg kg⁻¹ of body weight. The activity of peptides RYLGY and AYFYPEL in SHR was similar to that found for tripeptide VPP when orally administered at the same dose.     

α-Galactosidase and proteolytic activities of selected probiotic and dairy cultures in fermented soymilk

The metabolic activities of Lactobacillus acidophilus (LAFTI^® L10 and La4962) Bifidobacterium (lactis LAFTI^® B94 and longum Bl536), Lactobacillus casei (LAFTI^® L26 and Lc279), Lactobacillus delbrueckii ssp. bulgaricus Lb1466 and Streptococuss thermophilus St1342 were assessed in soymilk. Strains were initially analyzed for α-galactosidase activity and organic acid production in MRS broth at 37 °C. Consequently, soymilk was fermented with each strain and cell growth, production of organic acid, metabolism of oligosaccharides and proteolytic and ACE-inhibitory activities were assessed during 48 h of incubation at 42 °C. All strains exhibited variable α-galactosidase activity, with Bifidobacterium lactis B94 showing the highest activity. The oligosaccharide metabolism depended on α-galactosidase activity. B. lactis B94, S. thermophilus St1342 and L. acidophilus La4962 reduced raffinose substantially by 77.4%, 64.5% and 55.9%, respectively. All strains reached the desired therapeutic level of 10⁸ cfu/ml in soymilk after 48 h at 42 °C. The hydrolysis of protein in soymilk likely depended on strain (P < 0.0001) and time (P < 0.0001). The strains also released bioactive peptides with ACE-inhibitory activities between 17% and 43%.     

Multivariate analysis of proteolysis patterns differentiated the impact of six strains of probiotic bacteria on a semi-hard cheese

The individual contribution of 6 strains of probiotic bacteria (3 of Lactobacillus acidophilus and 3 of the Lactobacillus casei group) to proteolysis patterns in a semi-hard cheese was assessed. Control cheeses (without probiotics) and 2 types of experimental cheeses (with the addition of probiotics either directly to milk or by a 2-step fermentation method) were manufactured. Cheeses containing Lb. acidophilus showed the most extensive peptidolysis, which was evidenced by changes in the peptide profiles and a noticeable increase of free amino acids compared with control cheeses. The strains of the Lb. casei group showed a lower contribution to cheese peptidolysis, which consisted mainly of free amino acid increase. Two-step fermentation improved peptidolytic activity for only one of the cultures of Lb. acidophilus tested. The addition of Lb. acidophilus strains into cheese may be suitable not only for their beneficial health effect but also for their influence on secondary proteolysis, consistent with acceleration of ripening and improved flavor formation.     

Effect of acidification on the activity of probiotics in yoghurt during cold storage

The viability of Lactobacillus acidophilus LAFTI^® L10, Bifidobacterium lactis LAFTI^® B94, and L. paracasei LAFTI^® L26 and their proteolytic activities were assessed in yoghurt at different termination pH of 4.45, 4.50, 4.55, and 4.60 in the presence of L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342 during 28 days of storage at 4 °C. All strains achieved the recommended level of 6.00 log cfu g⁻¹ of the product with L. acidophilus LAFTI^® L10 and L. paracasei LAFTI^® L26 exceeding the number to 8.00 and 7.00 log cfu g⁻¹, respectively. Lactobacilli strains showed a good cellular stability maintaining constant concentration throughout storage period regardless of termination pH. On the other hand, the cell counts of B. lactis LAFTI^® B94 decreased by one log cycle at the end of storage. The presence of probiotic organisms enhanced proteolysis significantly in comparison with the control batch containing L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St1342 only. The proteolytic activity varied due to termination pH, but also appeared to be strain related. The increased proteolysis improved survival of L. delbrueckii ssp. bulgaricus Lb1466 during storage resulting in lowering of pH and production of higher levels of organic acids, which might have caused the low cell counts for B. lactis LAFTI^® B94.     

Identification of ACE-inhibitory peptides in different Spanish cheeses by tandem mass spectrometry

Different Spanish cheeses (Cabrales, Idiazábal, Roncal, Manchego, Mahón and a goat's milk cheese), made with diverse technologies and milk from different species, were studied for their angiotensin converting enzyme (ACE)-inhibitory activity. Among the water-soluble extract WSE<1000-Da of the different samples, Cabrales cheese was the most active with an inhibitory activity of 76.1%. On the contrary, Mahón cheese showed the lowest ACE-inhibitory index (56.6%). In order to identify the peptides involved in this activity, the WSE<1000-Da were analyzed by HPLC-MS/MS and off-line MS/MS. A total of 41 major peptides were identified in the different cheeses. Most of them derived from β-and α_s1-casein. Several of these peptides were selected on the basis of the presence of proline as the C-terminal amino acid, and they were chemically synthesized. All peptides showed moderate or low ACE-inhibitory activity. Peptide DKIHP [β-CN f(47–51)], a sequence detected in all samples except Mahón cheese, showed the highest inhibitory activity with an IC₅₀ value of 113.1 μM.     

Repair and growth of heat- and freeze-injured Escherichia coli O157:H7 in selective enrichment broths

Two Escherichia coli O157:H7 strains, ATCC 35150 and 43894, were heat injured in a beef infusion at 53°C for 40 and 50 min, respectively (1· 5–2·0 log₁₀cfu ml⁻¹of injury) and freeze injured at −25°C for 30 days (1 log₁₀cfu ml⁻¹of injury) as determined by plating on MacConkey agar with 0·60% bile salts #3 (Mac-BS) as the selective medium and on Brain Heart Infusion agar (BHIA) as the non-selective medium. Repair of injury was measured in five selective enrichment broths [buffered peptone water supplemented with vancomycin, cefsulodin, and cefixime (BPW-VCC), modified EC broth with novobiocin (mEC+n), enterohaemorrhagic E. coli enrichment broth (EEB), double modified TSB (dmTSB), and BCM^®E. coli enrichment broth (BCM^®-EB)] versus TSB as the non-selective control broth over 3 h incubation at 37°C and 42°C. Repair was measured as the increase in cfu ml⁻¹enumerated on Mac-BS with time vs the total cfu ml⁻¹(injured and uninjured cells) enumerated on BHIA. In mEC+n, EEB, and dmTSB some death of both heat- and freeze-injured cells occurred immediately during the 3 h incubation (decrease on BHIA plates), and there was either minimal or no repair of the injured cells at both temperatures. Efficient repair of heat injury was obtained with both BPW-VCC and BCM^®-EB, but the latter produced a growth rate and final cell concentration closer to TSB. In freeze-injury repair however, BPW-VCC gave poor results while repair in BCM^®-EB was equal to TSB. Both BCM^®-EB and BPW-VCC inhibited the growth of all Gram-positive and a select number of Gram-negative bacteria tested. The ability of the selective enrichment broth BCM^®-EB to resuscitate heat- and freeze-injured E. coli O157:H7 efficiently within 3 h, warrants further testing with other types of stress in both artificially and naturally contaminated foods.     

Assessing the proteolytic and lipolytic activities of single strains of mesophilic lactobacilli as adjunct cultures using a Caciotta cheese model system

Seventeen strains of mesophilic lactic acid bacteria, isolated from cheese (non-starter lactic acid bacteria, NSLAB) or sourdough, were used individually as adjunct cultures in a Caciotta cheese model system. Adjunct cultures were monitored by randomly amplified polymorphic DNA analysis and their cell counts mainly varied from ca. 9.0 to 8.0 log cfu g⁻¹ throughout 36 days of ripening. Adjunct cultures influenced differently cheese proteolysis. Both NSLAB and sourdough strains caused an extensive secondary proteolysis; however, some NSLAB strains produced the highest concentration of free amino acids. Principal component analysis (PCA) differentiated cheeses manufactured with NSLAB strains Lactobacillus parabuckneri B₉F_ST, Lb. paracasei B₆₁F₅, Lb. curvatus 2768 and Lb. rhamnosus ATCC 7469 based on the accumulation of Lys, Glu, Phe, Hist, Asp and Met. Assessment of cheese lipolysis showed that: (i) highest concentrations of free fatty acids (FFA) were found with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 (ca. 10 500 mg kg⁻¹); (ii) PCA differentiated cheeses manufactured with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 based on the accumulation of palmitic (C16:0) and linoleic (C18:2) acids, and those with Lb. curvatus 2768 and Lb. parabuckneri B₉F_ST based on the high concentration of short chain FFA; (iii) the cheese made with sourdough strain Lb. sanfranciscensis CB1 had the highest levels of unsaturated FFA.     

Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas

The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n = 3), commercial milk-based infant formulas (n = 6) and experimental infant formulas (n = 3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5–176 mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16–297 nmol BCM7 were released from 800 ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156 °C × 6–9 s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs.     

Survival and activity of selected probiotic organisms in set-type yoghurt during cold storage

The growth and metabolism of two probiotic organisms (L. acidophilus LAFTI^® L10 and Lactobacillus casei LAFTI^® L26) and a regular yoghurt culture (L. delbrueckii ssp. bulgaricus Lb1466 and Streptococcus thermophilus St1342) were studied in yoghurt containing 0.5%, 1.0%, and 1.5% (w/v) of high amylose corn starch powder (Hi-maize^®) or inulin. Viable cell counts of probiotic organisms, their metabolites and proteolytic activities, and viscosity of the yoghurts were determined during refrigerated storage for 28 d at 4 ^oC. In the presence of inulin, cultures showed better retention of viability (8.0 log cfu g⁻¹) in comparison with that of Hi-maize, which had a reduction by one log cycle. Lower concentrations of 0.5–1.0% Hi-maize improved (P<0.05) the production of propionic acid and also increased proteolytic activity of probiotic organisms substantially. A greater release of free amino acids may have sustained better growth of the organisms in yoghurts. Supplementation with either Hi-maize or inulin increased the viscosity of probiotic yoghurts significantly (P<0.05).     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Preparation and properties of probiotic concentrated yoghurt (labneh) fortified with conjugated linoleic acid

Milk of high conjugated linoleic acid (CLA) level (1.25 g per 100 g milk fat) was produced by inclusion of fish oil and rousted soy bean in the ration of Holstein cows as compared to 0.55 g per 100 g milk fat in the milk of animals receiving control diet. Milk of normal (control) and high CLA content (treatment) was spray‐dried. Labneh was made from 20 g L^?1 reconstituted milk using 3 mL per 100 mL yoghurt starter and 2 mL per 100 mL of probiotic cultures of Lactobacillus casei or Lactobacillus acidophilus. The control (C) and high CLA (T) labneh were analysed chemically and microbiologically, and their viscosities were determined during cold storage for 15 days. The fat content of labneh of high CLA was less than that of the control, but the total solids (TS) were unaffected by the CLA level. Labneh made with Lb. acidophilus had lower TS and higher acidity, exopolysaccharides and acetaldehyde contents and viscosity than that made with the use of Lb. casei. Labneh from the different treatments retained high counts of the added probiotic (>10⁸ cfu g^?1) throughout the storage period. The storage period had significant effects on all parameters determined.     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Growth and survival of Escherichia coli O157:H7 during the fermentation and storage of diluted cultured milk drink

The fate of various Escherichia coli O157:H7 strains including 933, A8993-C32, MF6707, 18731A, EK250 and EF304 during the fermentation and storage of diluted cultured milk drink fermented with Lactobacillus casei ssp.casei CCRC 11197 or L. delbrueckii ssp. bulgaricus CCRC 14009 were investigated in this study. E. coli O157:H7, regardless of strains, grew rapidly in skim milk and reached a maximum population of c. 8·0–9·0 log cfu ml⁻¹after c. 24 h of cultivation in mixed cultures with L. casei ssp. casei or L. delbrueckii ssp. bulgaricus. However, the population of E. coli O157:H7, depending on the strain and the lactic acid bacteria present, declined as cultivation proceeded further. With the inoculation of c. 5·0 log cfu ml⁻¹E. coli O157:H7, viable cells of this pathogen reduced to non-detectable level in the non-sugar-added cultured drink (pH 3·5) prepared with L. delbrueckii ssp.bulgaricus after one day storage at 7°C. Depending on the strains, E. coli O157:H7 survived in the non-sugar-added cultured drink prepared with L. casei ssp. casei for a period of <1–4 days. Adding sugar to cultured drink extended the survival period of E. coli O157:H7. The extent of the sugar-protective effect varied with different strains of E. coli O157:H7 and the amount of sugar added to the drink.     

Peptide profiles and angiotensin-I-converting enzyme inhibitory activity of fermented milk products: Effect of bacterial strain,fermentation pH,and storage time

Milk was fermented to defined pH values with 13 strains of lactic acid bacteria. The products were evaluated after 1 and 7 days of cold storage, and major peptides in selected products were identified. The Streptococcus thermophilus and Lactobacillus acidophilus strains used did not give rise to products with significant angiotensin-1-converting enzyme (ACE)-inhibition. The four Lactococcus lactis strains behaved similarly in fermentation, proteolysis and ACE-inhibition. The products made with the seven Lactobacillus helveticus strains varied. The highest ACE-inhibitory activity was obtained with two highly proteolytic strains of Lb. helveticus and with the Lactococcus strains. Fermentation from pH 4.6 to 4.3 with these strains slightly increased the ACE-inhibitory activity, whilst fermentation to pH 3.5 with Lb. helveticus reduced the ACE-inhibitory activity. Cold storage dramatically increased the ACE-inhibitory activity of some products. A non-linear correlation was found between peptide amount and ACE-inhibitory activity, and peptides contributing to the ACE-inhibitory activity were identified.     

Survival kinetics of Ephestia kuehniella eggs during 46–75 °C heat treatment

The effect of heat treatment on the survival of Ephestia kuehniella eggs was examined. Samples of 60 eggs were immersed in hot water at constant temperature in the 46–75 °C range for 5–1200 s. Following heat treatment and cooling, the eggs were stored at 24 ± 1 °C in a growth chamber for 7 days before survival evaluation. Statistical analysis of the data demonstrated that the thermal survival kinetics were best represented by a first-order reaction. The rate constant had an Arrhenius-type dependence over the 54–75 °C temperature range. Kinetic parameters were estimated by non-linear regression. The activation energy (E_a) and rate constant (k_ref) at the reference temperature (T_ref = 64.8 °C), were determined as 102.2 ± 6.2 kJ mol⁻¹ and 0.061 ± 0.003 s⁻¹, respectively, over the 54–75 °C temperature range. A 0.01% survival rate was obtained after 50 s at 75 °C. The data at temperatures below 50 °C were not in accordance with those at higher temperatures. Above this temperature, mortality was likely due to physiological disorders, as noted on a DSC thermogram.