Application of an active alginate coating to control the growth of Listeria monocytogenes on poached and deli turkey products

The relatively high prevalence of Listeria monocytogenes in ready-to-eat (RTE) turkey products is of great concern. The overall objective of this study was to develop antimicrobial edible coating formulations to effectively control the growth of this pathogen. The antimicrobials studied were nisin (500 IU/g), Novagard CB 1 (0.25%), Guardian NR100 (500 ppm), sodium lactate (SL, 2.4%), sodium diacetate (SD, 0.25%), and potassium sorbate (PS, 0.3%). These were incorporated alone or in binary combinations into five edible coatings: alginate, κ-carrageenan, pectin, xanthan gum, and starch. The coatings were applied onto the surface of home-style poached and processed deli turkey discs inoculated with ~ 3 log CFU/g of L. monocytogenes. The turkey samples were then stored at 22 °C for 7 days. For poached and processed deli turkey, the coatings were found to be equally effective, with pectin being slightly less effective than the others. The most effective poached turkey treatments seemed to be SL (2.4%)/SD (0.25%) and Nisin (500 IU/g)/SL (2.4%), which yielded final populations of 3.0 and 4.9 log CFU/g respectively compared to the control which was 7.9 log CFU/g. For processed deli turkey, the most effective antimicrobial treatments seemed to be Nisin (500 IU/g)/SD (0.25%) and Nisin (500 IU/g)/SL (2.4%) with final populations of 1.5 and 1.7 log CFU/g respectively compared to the control which was 6.5 log CFU/g. In the second phase of the study, home-style poached and store-purchased roasted (deli) turkey inoculated with the pathogen at a level of ~ 3 log CFU/g were coated with alginate incorporating selected antimicrobial combinations and stored for 8 weeks at 4 °C. Alginate coatings supplemented with SL (2.4%)/PS (0.3%) delayed the growth of L. monocytogenes with final counts reaching 4.3 log CFU/g (home-style poached turkey) and 6.5 log CFU/g (roasted deli turkey) respectively while the counts in their untreated counterparts were significantly higher (P < 0.05) reaching 9.9 and 7.9 log CFU/g, respectively. This study therefore demonstrates the effectiveness of using alginate-based antimicrobial coatings to enhance the microbiological safety and quality of RTE poultry products during chilled storage.     

Histamine production by Enterobacter aerogenes in sailfish and milkfish at various storage temperatures

Enterobacter aerogenes was studied for its growth and ability to promote the formation of total volatile base nitrogen (TVBN) and histamine in sailfish (Istiophorus platypterus) and milkfish (Chanos chanos) stored at various temperatures from -20 to 37 degrees C. The optimal temperature for bacterial growth in both fish species was 25 degrees C, whereas the optimal temperature for histamine formation was 37 degrees C. The two fish species inoculated with E. aerogenes, when not properly stored at low temperatures such as 15 degrees C for 36 h, formed histamine at above the U.S. Food and Drug Administration hazardous guideline level of 50 mg/100 g. Milkfish was a better substrate than sailfish for histamine formation by bacterial histidine decarboxylation at elevated temperatures (> 15 degrees C). Although higher contents of TVBN were detected in the spiked sailfish than milkfish during the same storage time at temperatures above 15 degrees C, the use of the 30-mg/100 g level of TVBN as a determination index for fish quality and decomposition was not a good criterion for assessing potential histamine hazard for both fish species. Bacterial growth was controlled by cold storage of the fish at 4 degrees C or below, but histamine formation was stopped only by frozen storage. Once the frozen fish samples were thawed and stored at 25 degrees C, histamine started to accumulate rapidly and reached levels greater than the hazardous action level in 36 h.     

Fate of post-processing inoculated Listeria monocytogenes on vacuum-packaged pepperoni stored at 4, 12 or 25 °C

If present, Listeria monocytogenes may not be eliminated during processing of pepperoni or may be introduced during peeling, slicing, or packaging. We evaluated the fate of the pathogen on sliced inoculated pepperoni during vacuum-packaged storage, and potential differences in survival among three types of inocula, including nonacid-adapted, acid-adapted and pepperoni extract-habituated cultures. Commercial pepperoni (two replicates, three samples per treatment) was sliced and inoculated (3 to 4 log CFU/cm²), before vacuum-packaging and storage for up to 180 days at 4, 12 or 25 °C. Samples were periodically analyzed for pathogen counts (PALCAM agar) and total bacterial counts (tryptic soy agar with 0.6% yeast extract). The pH of the product was relatively stable (4.50–4.81) throughout storage. Overall, levels of the pathogen (all inocula) and total counts decreased continuously during storage at all temperatures. The pathogen died slower at 4 °C than at 12 and 25 °C, while at 12 and 25 °C the death rates were similar. Death rates depended on type of inoculum and generally decreased in the order: acid-adapted, extract-habituated and nonacid-adapted inoculum. At day 60, pathogen levels were below the detection limit and remained undetectable throughout the rest of the 180-day storage period, regardless of inoculum type and storage temperature. Therefore, storage of sliced vacuum-packaged pepperoni, especially at ambient temperature, prior to consumption may reduce the potential risk of listeriosis.     

Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery

Illnesses from Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella have been associated with the consumption of numerous produce items. Little is known about the effect of consumer handling practices on the fate of these pathogens on celery. The objective of this study was to determine pathogen behavior at different temperatures under different storage conditions. Commercial fresh-cut celery was inoculated at ca. 3 log CFU/g onto either freshly cut or outer uncut surfaces and stored in either sealed polyethylene bags or closed containers. Samples were enumerated following storage for 0, 1, 3, 5, and 7 days when held at 4 °C or 12 °C, and after 0, 8, and 17 h, and 1, and 2 days when held at 22 °C. At 4 °C, all populations declined by 0.5–1.0 log CFU/g over 7 days. At 12 °C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased by ca. 0.5 log CFU/g over 7 days. At 22 °C, E. coli O157:H7, Salmonella, and L. monocytogenes populations increased by ca. 1, 2, or 0.3 log CFU/g, respectively, with the majority of growth occurring during the first 17 h. On occasion, populations on cut surfaces were significantly higher than those on uncut surfaces. Results indicate that populations are reduced under refrigeration, but survive and may grow at elevated temperatures.     

Biogenic amine changes in barramundi (Lates calcarifer) slices stored at 0 °C and 4 °C

The biogenic amines formation in barramundi (Lates calcarifer) slices kept for 15 days at 0 °C and 4 °C were investigated using nine biogenic amines, total plate counts and biogenic amines formers. Significant differences in biogenic amines concentrations of barramundi slices stored at 4 °C and at 0 °C after 3 days of storage were observed. All amines, except tryptamine, 2-phenylethylamine, tyramine and agmatine in the slices increased with time during storage at both temperatures. At the end of the storage period, histamine concentrations were 82 mg/kg and 275 mg/kg for samples kept at 0 °C and 4 °C, respectively. At day 15, the total plate count was approximately 8.6 log CFU/g for sample kept at 0 °C and 9.7 log CFU/g for samples kept at 4 °C. Histamine-forming bacteria (HFB) in all samples ranged from 5.4 to 6.1 log CFU/g at 0 °C and 4 °C, respectively. The observed shelf-life of barramundi slices were 6–9 days.     

Cooking quality of faba bean after storage at high temperature and the role of lignins and other phenolics in bean hardening

Selected physical and chemical characteristics of faba beans (Vicia faba L.) cv. Fiesta were studied after 12 months storage at 5, 15, 25, 37, 45 or 50 °C (±2 °C) in relation to the hard-to-cook phenomenon. In comparison with control (seeds stored at 5 °C), seeds stored at 15 and 25 °C demonstrated non-significant (p?0.05) changes in most of the physical and chemical characteristics including hydration and swelling coefficients, acid detergent fibre, lignin and tannin contents, whereas seeds stored at ?37 °C demonstrated significant changes (p?0.05). Solutes and electrolytes leaching after 18 h soaking substantially increased with increased temperature. Faba bean hardness tested by the hard-to-cook test also increased substantially with increased storage temperature. After 8 h soaking followed by 2 h cooking, the puncture force required for seeds stored at 5 °C was 3.3 N seed⁻¹ whereas seeds stored at 50 °C required a much higher puncture force of 15.2 N seed⁻¹. There was a high negative correlation (r²=0.98) between storage temperature and cooking ability of faba bean. Substantial increases in acid detergent fibre and lignin contents occurred with increased storage temperatures. There was a three-fold increase in lignin content of faba bean stored at 50 °C compared to those stored at 5 °C and it was correlated with bean hardness (r²=0.98). Storage at high temperatures for 12 months led to a substantial reduction in total free phenolics especially in the testa and there was a greater reduction with increasing storage temperature. Reduction in free phenolics was negatively correlated (r²=0.75) with bean hardness.     

Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing

Escherichia coli O157:H7 attached to beef-contact surfaces found in beef fabrication facilities may serve as a source of cross-contamination. This study evaluated E. coli O157:H7 attachment, survival and growth on food-contact surfaces under simulated beef processing conditions. Stainless steel and high-density polyethylene surfaces (2 × 5 cm) were individually suspended into each of three substrates inoculated (6 log CFU/ml or g) with E. coli O157:H7 (rifampicin-resistant, six-strain composite) and then incubated (168 h) statically at 4 or 15 °C. The three tested soiling substrates included sterile tryptic soy broth (TSB), unsterilized beef fat-lean tissue (1:1 [wt/wt]) homogenate (10% [wt/wt] with sterile distilled water) and unsterilized ground beef. Initial adherence/attachment of E. coli O157:H7 (0.9 to 2.9 log CFU/cm²) on stainless steel and high-density polyethylene was not affected by the type of food-contact surface but was greater (p < 0.05) through ground beef. Adherent and suspended E. coli O157:H7 counts increased during storage at 15 °C (168 h) by 2.2 to 5.4 log CFU/cm² and 1.0 to 2.8 log CFU/ml or g, respectively. At 4 °C (168 h), although pathogen levels decreased slightly in the substrates, numbers of adherent cells remained constant on coupons in ground beef (2.4 to 2.5 log CFU/cm²) and increased on coupons in TSB and fat-lean tissue homogenate by 0.9 to 1.0 and 1.7 to 2.0 log CFU/cm², respectively, suggesting further cell attachment. The results of this study indicate that E. coli O157:H7 attachment to beef-contact surfaces was influenced by the type of soiling substrate and temperature. Notably, attachment occurred not only at a temperature representative of beef fabrication areas during non-production hours (15 °C), but also during cold storage (4 °C) temperatures, thus, rendering the design of more effective sanitation programs necessary.     

Combined antimicrobial effect of oregano essential oil and caprylic acid in minced beef

Oregano essential oil (OEO) and caprylic acid (CA) are highly aromatic natural antimicrobials with limited individual application in food. We proved their combined additive effect when used in meat. Application of 0.5% CA and 0.2% OEO (v/w) with 0.1% of citric acid in vacuum packed minced beef inoculated with Listeria monocytogenes at a concentration of 5 log cells/g reduced counts of lactic acid bacteria by 1.5 log CFU/g and counts of psychrotrophic bacteria and L. monocytogenes by more than 2.5 log CFU/g at the end of storage at 3 °C for 10 days. In sensory evaluation the samples with OEO showed during the whole experiment statistically better scores than control, whereas the samples treated with CA showed worse colour attributes.     

Histamine Formation and Bacterial Spoilage of Albacore Harvested off the U.S. Northwest Coast

Iced and previously frozen albacore were monitored for histamine formation and bacterial growth during storage at 0–37°C. The optimum temperature for histamine formation in albacore tuna (Thunnus alalunga) was 25°C, and whole fish were more susceptible to histamine formation than dressed fish at that temperature. Storage at 25°C resulted in the highest histamine level, 60.4 mg/100g in whole fish stored for 7 days. When albacore were frozen prior to storage, reduced amount of histamine was found at 7.14 mg/100 g after 7 day storage at 25°C, only after decomposition became obvious. No histamine was found in any of the albacore samples stored in ice for 18 days.     

Efficacy of washing with hydrogen peroxide followed by aerosolized antimicrobials as a novel sanitizing process to inactivate Escherichia coli O157:H7 on baby spinach

Aerosolization was investigated as a potential way to apply allyl isothiocyanate (AIT), hydrogen peroxide (H₂O₂), acetic acid (AA) and lactic acid (LA) on fresh baby spinach to control Escherichia coli O157:H7 during refrigeration storage. In this study, baby spinach leaves were dip-inoculated with E. coli O157:H7 to a level of 6 log CFU/g and stored at 4 °C for 24 h before treatment. Antimicrobials were atomized into fog-like micro-particles by an ultrasonic nebulizer and routed into a jar and a scale-up model system where samples were treated. Samples were stored at 4 °C for up to 10 days before the survival of the cells was determined. A 2-min treatment with 5% AIT resulted in a > 5-log reduction of E. coli O157:H7 on spinach after 2 days refrigeration regardless if the samples were pre-washed or not; however, this treatment impaired the sensory quality of leaves. Addition of LA to AIT improved the antimicrobial efficacy of AIT. In the jar system, washing with 3% H₂O₂ followed by a 2-min treatment of 2.5% LA + 1% AIT or 2.5% LA + 2% AIT reduced E. coli O157:H7 population by 4.7 and > 5 log CFU/g, respectively, after 10 days refrigeration. In the scale-up system, up to 4-log reduction of bacterial population was achieved for the same treatments without causing noticeable adverse effect on the appearance of leaves. Thus, this study demonstrates the potential of aerosolized AIT + LA as a new post-washing intervention strategy to control E. coli O157:H7 on baby spinach during refrigeration storage.     

Survival,fermentation activity and storage stability of spray dried Lactococcus lactis produced via different atomization regimes

Dried powders containing Lactococcus lactis ssp. cremoris were produced using laboratory and pilot scale spray dryers with lactose:whey protein isolate (3:1) as a protective medium. The effects of storage temperature (25, 4 and −18 °C) and time (30, 60 and 90 days) were studied. The survival and fermentation activity of the dried bacterial cells were significantly lower when the powders were stored at 25 °C compared to those stored at 4 and −18 °C; powders stored at 4 and −18 °C were statistically similar. The survival and fermentation activity of bacterial cells obtained from a laboratory scale two-fluid nozzle spray dryer were found to be higher than those of cells obtained from a pilot scale two-fluid spray dryer. A rotary wheel atomizer gave significantly higher survival and activity in the same dryer. These observations are consistent with cell damage due to high characteristic shear rates in the atomization process in nozzle type atomizers. The presence of ascorbic acid (oxygen scavenger) in the powder composition was found to improve both the survival and the maintenance of fermentation activity of the dried bacterial cells significantly during storage. The survival and fermentation activity of dried bacterial cells in stored powders indicated that these parameters are system-specific and can be strongly affected by the storage temperature and presence or absence of antioxidant, and also by upstream processing conditions such as the mode of atomization and presence or absence of antioxidants in the dryer feed.     

Cultured C2C12 cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells

The mechanisms of bacterial attachment to meat tissues need to be understood to enhance meat safety interventions. However, little is known about attachment of foodborne pathogens to meat muscle cells. In this study, attachment of six Escherichia coli and two Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C₂C₁₂, was measured, including the effect of temperature. At 37 °C, all but one strain (EC623) attached to C₂C₁₂ cells, whereas only five of eight strains (M23Sr, H10407, EC473, Sal1729a and Sal691) attached to primary cells. At 10 °C, two strains (H10407 and EC473) attached to C₂C₁₂ cells, compared to four strains (M23Sr, EC614, H10407 and Sal1729a) of primary cells. Comparing all strains at both temperatures, EC614 displayed the highest CFU per C₂C₁₂ cell (4.60 ± 2.02 CFU/muscle cell at 37 °C), whereas greater numbers of M23Sr attached per primary cell (51.88 ± 39.43 CFU/muscle cell at 37 °C). This study indicates that primary bovine muscle cells may provide a more relevant model system to study bacterial attachment to beef carcasses compared to cell lines such as C₂C₁₂.     

Histamine formation by histamine-forming bacteria and yeast in mustard pickle products in Taiwan

The occurrence of histamine, histamine-forming bacteria and yeast were tested in 37 mustard pickle products sold in both retail markets and supermarkets in southern Taiwan. Aerobic plate count (APC), total coliform, and Escherichia coli were also tested for microbiological quality. Salt content, pH value, titratable acidity and sulphite content were determined for quality of mustard pickle products. Only one retail market sample and one supermarket sample had 8.9 and 7.4 mg histamine per 100 g products, although the average content for each of the nine biogenic amines was less than 2 mg/100 g. Ten histamine-forming bacterial strains and 6 histamine-producing yeast strains capable of producing 8.7 to 1260 ppm of histamine in trypticase soy broth (TSB) supplemented with 1% l-histidine (TSBH) were identified as Staphylococcus capitis (four strains),Staphylococcus pasteuri (two strains), Enterobacter cloacae (four strains), Candida glabrata (two strains) and Candida rugosa (four strains). S. capitis, which was previously reported to be halotolerant, was a potent histamine-former, capable of producing more than 1000 ppm of histamine in TSBH in the presence of 0.5–10% NaCl. The numbers of the aerobic plate count (APC) in all samples were below the Taiwanese regulatory level of 5 log CFU/g. None of the samples contained total coliform or E. coli. The values of pH, salt content, titratable acidity and sulphite content in all samples ranged from 3.8% to 5.0%, 2.0% to 10.0%, 0.21% to 1.18% and <2.0–1876 ppm, respectively.     

The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.     

Validation of a stochastic modelling approach for Listeria monocytogenes growth in refrigerated foods

A stochastic modelling approach was developed to describe the distribution of Listeria monocytogenes contamination in foods throughout their shelf life. This model was designed to include the main sources of variability leading to a scattering of natural contaminations observed in food portions: the variability of the initial contamination, the variability of the biological parameters such as cardinal values and growth parameters, the variability of individual cell behaviours, the variability of pH and water activity of food as well as portion size, and the variability of storage temperatures. Simulated distributions of contamination were compared to observed distributions obtained on 5 day-old and 11 day-old cheese curd surfaces artificially contaminated with between 10 and 80 stressed cells and stored at 14 °C, to a distribution observed in cold smoked salmon artificially contaminated with approximately 13 stressed cells and stored at 8 °C, and to contaminations observed in naturally contaminated batches of smoked salmon processed by 10 manufacturers and stored for 10 days a 4 °C and then for 20 days at 8 °C. The variability of simulated contaminations was close to that observed for artificially and naturally contaminated foods leading to simulated statistical distributions properly describing the observed distributions. This model seems relevant to take into consideration the natural variability of processes governing the microbial behaviour in foods and is an effective approach to assess, for instance, the probability to exceed a critical threshold during the storage of foods like the limit of 100 CFU/g in the case of L. monocytogenes.     

Stability of lipids and fatty acids in frozen and gamma irradiated Patagonian toothfish (Dissostichus eleginoides) from the Southwestern Atlantic

Patagonian toothfish were captured in the Southwestern Atlantic Ocean (FAO Zone N° 41). The fatty acid profile of total lipids and the triacylglycerol and phospholipid content of control and irradiated samples (1 and 5 kGy) stored at −18 °C were analyzed at 0 and 293 days post irradiation. The fatty acids are mainly monounsaturated acids (47 g/100 g total fatty acids), the most abundant one being oleic acid (18:1 n-9). This is followed in order of abundance by saturated fatty acids (26 g/100 g total fatty acids), consisting mainly of palmitic acid (16:0). Polyunsaturated fatty acids were less abundant (17 g/100 g total fatty acids) and consisted mainly of eicosapentaeonic (20:5 n-3) and docosahexaenoic (22:6 n-3) acids. Triacylglycerol content was 563.07 mg/mL oil, whereas phospholipids were 11.21 mg/mL oil. Gamma irradiation did not significantly affect the fatty acid profile or triacylglycerol and phospholipid content of P. toothfish stored for 293 days at −18 °C. The results suggest that the species exhibits a marked stability when subjected to irradiation and prolonged storage in the frozen state.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48

The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 μg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 °C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 μg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 μg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus.     

Post-mortem changes in viscera of cuttlefish Sepia officinalis L. during storage at two different temperatures

We observed the post-mortem changes in viscera of cuttlefish Sepia officinalis in order to apply the data to autolysate production. Cuttlefish viscera were stored at 4 or 25 °C and sampled regularly over 4 months. Our results showed that total acid proteases and cathepsins were rapidly released to the extracellular medium due to the breakdown of lysosomes. Total alkaline protease activity increased 2 h after death due to the breakdown of zymogene vesicles. The same patterns were found with trypsin, chymotrypsin and α-amylase activities. After 10 days of incubation, no endogenous enzymatic activity was found. After 50 days of storage, the TCA soluble protein levels decreased rapidly to approximately 30% due to protein degradation and aggregation. After 10 days, the pH of viscera stored at 25 °C was alkaline, whereas in the viscera stored at 4 °C the pH increased more slowly. As significant reduction in the protein molecular weight due to autolysis, was also observed.