Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Effect of Penicillium roqueforti and incorporation of whey cheese on volatile profiles and sensory characteristics of mould‐ripened Civil cheese

In this study, four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as secondary starter for the manufacture of mould‐ripened Civil cheese with and without addition of the whey cheese Lor; in parallel, secondary starter‐free counterparts were manufactured. A total of 83 compounds were identified. Ketones, alcohols and esters were the principal classes of volatile components. Principal component analysis of the headspace volatiles grouped cheeses by age and type. P. roqueforti inoculated cheese was clearly separated from the other cheeses at 180 days of ripening, and these cheeses were characterised with high levels of ketones (e.g., 2‐butanone, 2‐heptanone). Differences in the panel scores between the cheese samples were not significant during the first stage of ripening (up to 60 days); as ripening proceeded, these differences were become evident and P. roqueforti inoculated cheeses received higher scores than others. Addition of Lor in the manufacture of mould‐ripened Civil cheese caused lower points by the sensory panel, and the cheese inoculated with P. roqueforti and Lor‐free was the best type of mould‐ripened Civil cheese. The results showed that the use of P. roqueforti in the manufacture of mould‐ripened Civil cheese has significant impact on the volatile profiles and sensory attributes.     

Production and partial purification of proteases from Aspergillus oryzae grown in a medium based on whey protein as an exclusive nitrogen source

Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of β-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.     

Biotechnological characterisation of exocellular proteases produced by enological Hanseniaspora isolates

Twenty‐six enological Hanseniaspora isolates were identified by ITS PCR‐RFLP and D1/D2 domain of 26S rDNA sequencing and assayed for exocellular protease production. Based on qualitative data, six isolates, belonging to H. guilliermondii, H. valbyensis and H. occidentalis species, were selected to continue the study. Analytical procedure was optimised, and protease activities were quantified and characterised on the basis of different biotechnological factors. Protease activity was quite glucose, fructose and ethanol content independent, but divalent cation affects activity; these data support that they were aspartic proteases. The effect of 2‐mercaptoethanol suggests the importance of disulphide bonds to maintain the structure of the active centre. Our results show these enzymes could be suitable for using in biotechnological processes at neutral pH, such as cheese, bread and meat industries. This is, to our knowledge, the first work showing the induction process to induce and characterise proteolytic activity in Hanseniaspora isolates.     

Effect of nisin‐producing Lactococcus lactis starter cultures on the inhibition of two pathogens in ripened cheeses

The effect of Lactococcus lactis nisin‐producing strains, isolated from Italian fermented foods, on the survival of two foodborne pathogens namely Listeria monocytogenes and Staphylococcus aureus was investigated in experimental cheese production. One of the three Lactobacillus lactis nisin innoculated as starters, Lactobacillus lactis 41FL1 lowered S. aureus count by 1.73 log colony‐forming units (cfu)/g within the first 3 days, reaching the highest reduction, 3.54 log cfu/g, by the end of ripening period of 60 days. There was no effect on L. monocytogenes. The application of L. lactis 41FL1 as bioprotective culture in controlling S. aureus shows considerable promise.     

Production of Lipase from Candida rugosa Using Cheese Whey through Experimental Design and Surface Response Methodology

The present work investigates the production of lipases from Candida rugosa in a culture medium containing cheese whey and also determines the importance of the components of the used culture medium using two experimental design and surface response methodology. When pure cheese whey was used as culture medium, the lipolytic activity measured in the broth, after 120 h of fermentation, was 5.18 U/mL. From the first experimental design, it was possible to conclude that the combination of brewery co-product, yeast and malt extract, Tween 80, and olive oil with cheese whey provided an average increase of 15 U/mL in the enzymatic activity, which represented 287.5% in relation to the activity obtained using only with cheese whey as substrate. Nevertheless, this essay did not provide a predictive model for the lipase production using the studied components. From the second experimental design, with suppression of yeast and malt extract, the best values of enzymatic activity were, on average, 28% lesser than the observed in the first optimization, but yet 180% higher than the obtained values when only cheese whey was present as substrate. A second-order model correlating the used components could be achieved, indicating that high concentrations of brewery co-product and Tween 80 enhanced the production of C. rugosa lipases in a medium incorporating cheese whey, which, at high concentrations, can substitute olive oil that may have its concentration diminished in this condition.     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.     

Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Growth and enterotoxin production of Staphylococcus aureus in Iranian ultra‐filtered white cheese

This study aimed to investigate the growth and enterotoxin production of Staphylococcus aureus during the ripening and storage of Iranian ultra‐filtered white cheese (IUFWC). According to the results, the addition of starter culture had significant inhibitory effects on the growth and enterotoxin production of S. aureus. Moreover, prolong survival and enterotoxin production were observed in the samples with the highest inoculum (5 log CFU/g) of S. aureus. It was concluded that proper activity of the starter culture together with the refrigeration storage of IUFWC was the key element in inhibiting the growth and enterotoxin production of S. aureus.     

PS0312 菌株发酵产淀粉酶及温度与pH 值对酶活力的影响

PS0312 菌株是具有特殊生物学特性的青霉菌株。以三角瓶固体培养研究不同条件与菌株产淀粉酶的关系,以及温度与pH 值对酶活力的影响。结果表明：PS0312 菌株以麸皮30.04%、大豆饼粉3.70%、谷壳3.70% 及55.56% 蒸馏水组成的培养基固体发酵湿麸曲淀粉酶产量最高。单因子试验结果表明：以培养基pH3、108 个/mL的种子液接种量1.85%、培养温度28℃、培养时间96h 产淀粉酶量最大。PS0312 菌株产淀粉酶在pH2.0～10.0 内具较高活性,酶活大小与pH 值的关系成双峰形;pH3 的条件下酶活性最高,相对酶活100%;pH9 的条件下酶活次之,相对酶活84.98%。酶反应最适温度为60℃,90℃条件下相对酶活为39.06%。研究表明：PS0312 菌株发酵产淀粉酶是一种在强酸、强碱条件下都具有较高的酶活性和耐高温的特殊的酸性、中温淀粉酶,可在较宽的温度与pH 值范围下应用。     

Milk‐clotting enzymes produced by Aspergillus flavo furcatis strains on Amazonic fruit waste

Six strains of Aspergillus flavo furcatis were screened to investigate milk‐clotting enzyme production by fermentation in natural liquid medium. The growth media comprised extracts of cupuaçu exocarp+rice bran [10% or 20% (v/v) CE+RB] and açai waste+rice bran [10% or 20% (v/v) AW+RB] with or without supplementation of 0.1% (w/v) yeast extract and 0.5% (w/v) gelatin. Significant values of milk‐clotting activity were determined by A. flavo furcatis DPUA 1461 and DPUA 1608, in the standard and natural media, respectively. According to criteria of clot and whey formations, 8.3% of the samples tested were classified as strong coagulation, 41.70% showed weak coagulation and in 50% was not observed milk coagulation. The enzyme optimal action of DPUA 1608, the selected strain, was at 40 °C and pH 7.0. Milk‐clotting proteases were inhibited by pepstatin (94.72%) and moderately inhibited by the others metal ions tested.     

Proteolytic and angiotensin‐converting enzyme‐inhibitory activities of selected probiotic bacteria

This study was carried out to examine the proteolytic and angiotensin‐converting enzyme (ACE‐I) activities of probiotic lactic acid bacteria (LAB) as influenced by the type of media, fermentation time, strain type and media supplementation with a proteolytic enzyme (Flavourzyme^®). Lactobacillus casei (Lc210), Bifidobacterium animalis ssp12 (Bb12), Lactobacillus delbrueckii subsp. bulgaricus (Lb11842) and Lactobacillus acidophilus (La2410) were grown in 12% of reconstituted skim milk (RSM) or 4% of whey protein concentrates (WPC‐35) with or without combination (0.14%) of Flavourzyme^® for 12 h at 37 °C. All the strains were able to grow in both media depending on type of strains used and fermentation time. All the strains showed higher proteolytic activity and produced more antihypersensitive peptides when grown in RSM medium at 12 h, when compared to WPC. Combination with Flavourzyme^® also increased LAB growth and proteolytic and ACE‐I activities. Of the four strains used, Bb12 and La2410 outperformed Lc210 and Lb11842. The highest ACE‐I activity and proteolytic activity were found in B. animalis ssp12 combined with Flavourzyme^®. Flavourzyme^® led to an increase in the production of bioactive peptides with ACE‐I activity during 12 h at 37 °C.     

Nisin treatments to control Listeria monocytogenes post-processing contamination on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages

Post-processing contamination and growth of Listeria monocytogenes in whey cheeses stored under refrigeration is an important safety concern. This study evaluated commercially available nisin (Nisaplin^®) as a biopreservative to control L. monocytogenes introduced post-processing on Anthotyros, a traditional Greek whey cheese, stored at 4°C in vacuum packages for up to 45 days. The whey used (pH 6.5–6.7) was from Feta cheese manufacture, and it was subjected either to natural acidification (pH 5.3, readjusted to 6.2 with 10% NaOH) prior to heating, or to direct acidification (pH 6.0–6.2) at 80°C with 10% citric acid. Nisin was added either to the whey (100 or 500 IU g⁻¹) prior to heating, or to the cheese (500 IU g⁻¹) prior to packaging, also inoculated with ca. 10⁴ cfu g⁻¹ of L. monocytogenes strain Scott A. In cheese samples without nisin, L. monocytogenes (PALCAM agar) exceeded 7 log cfu g⁻¹ after the first 10 days of storage, irrespective of the whey acidification method. All nisin treatments had an immediate lethal effect (0.7–2.2 log reduction) on L. monocytogenes populations at inoculation (day 0), which was more pronounced with 500 IU g⁻¹ added to the whey. This treatment also suppressed L. monocytogenes growth below the inoculation level for 30 and 45 days in naturally and directly acidified samples, respectively. All other treatments had weak antilisterial effects. Nisin reversed the natural spoilage flora of Anthotyros cheese from Gram-positive to Gram-negative, and this ecological alteration was far more pronounced in the most effective antilisterial treatments.     

Characterization of a new isolate of Lactobacillus fermentum IFO 3956 from Egyptian Ras cheese with proteolytic activity

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases.     

Production of a whey‐based functional beverage supplemented with soy isoflavones and phytosterols

Reconstituted whey beverages were prepared from whey powder by adding either soy isoflavones or phytosterols as functional compounds (at levels of 0%, 0.25%, 0.50% or 1.0% w/v) and probiotic bacteria (Lactobacillus acidophilus LA‐5 or Lactobacillus casei LBC‐81). The addition of nutraceuticals did not change the basic composition of the products. However, a time‐dependent increase in sedimentation/phase separation and acidity of fermented functional beverages was observed. Samples supplemented with phytosterols were more preferred by the panel group than the samples supplemented with isoflavones, and no considerable differences were noted between the control and phytosterol‐added samples in terms of overall perception.     

Mycotoxin production capability of Penicillium roqueforti in strains isolated from mould-ripened traditional Turkish civil cheese

Mould-ripened civil is a traditional cheese produced mainly in eastern Turkey. The cheese is produced with a mixture of civil and whey curd cheeses (lor). This mixture is pressed into goat skins or plastic bags and is ripened for more than three months. Naturally occurring moulds grow on the surface and inside of the cheese during ripening. In this research, 140 Penicillium roqueforti strains were isolated from 41 samples of mould-ripened civil cheese collected from Erzurum and around towns in eastern Turkey. All strains were capable of mycotoxin production and were analysed using an HPLC method. It was established that all the strains (albeit at very low levels) produced roquefortine C, penicillic acid, mycophenolic acid and patulin. The amounts of toxins were in the ranges 0.4–47.0, 0.2–43.6, 0.1–23.1 and 0.1–2.3 mg kg^?1, respectively. Patulin levels of the samples were lower than the others. The lowest level and highest total mycotoxin levels were determined as 1.2 and 70.1 mg kg^?1 respectively. The results of this preliminary study may help in the choice of secondary cultures for mould-ripened civil cheese and other mould-ripened cheeses.     

Viability of the Lactobacillus rhamnosus HN001 Probiotic Strain in Swiss‐ and Dutch‐Type Cheese and Cheese‐Like Products

The objective of this study was to determine the viability of the probiotic Lactobacillus rhamnosus HN001 in Swiss‐type and Dutch‐type cheese and cheese‐like products (milk fat is substituted by stearin fraction of palm fat) during manufacture, ripening, and storage. The use of the probiotic L. rhamnosus HN001 in Dutch‐type cheese and cheese‐like products significantly (P = 0.1) changed their chemical composition (protein and fat content) and an insignificant increase (approximately 1.6% in cheese‐like products and approximately 0.3% in cheese) in yield. L. rhamnosus HN001 did not affect the rate of changes in the pH of ripened cheese and cheese‐like products. A minor increase in probiotic counts was observed in initial stages of production and were partially removed with whey. Ripened cheese and cheese‐like products were characterized by high survival rates of probiotic bacteria which exceeded 8 log CFU/g after ripening. An insignificant reduction in the number of viable probiotic cells was noted during storage of Swiss‐type and Dutch‐type cheese, whereas a significant increase in probiotic cell counts was observed in cheese‐like products during storage.     

Proteolysis in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ŁOCK 0900 probiotic strain

Selected parameters related to proteolysis were evaluated in dry‐aged loins manufactured with sonicated pork and inoculated with Lactobacillus casei ?OCK 0900 probiotic strain. Dehydration, pH, water activity and total nitrogen content were not affected by ultrasound treatment. Neither sonication nor inoculation has an influence on L* and a* colour parameters. Sonication significantly influenced the pattern of proteolysis providing higher nonprotein nitrogen content (NPN) and proteolysis index (PI). The highest intensity of proteolysis, as assessed by NPN and PI, occurred in an inoculated sample. Neither sonication nor inoculation with L. casei had a significant effect on total viable counts. Loins inoculated with L. casei had the highest lactic acid bacteria count, followed by the sonicated and nonsonicated control. This research shows that sonication followed by inoculation with L. casei ?OCK 0900 could be used as an effective measure to speed up the proteolytic changes in dry‐aged meat cuts.     

Effects of Mentha longifolia L. essential oil and Lactobacillus casei on the organoleptic properties and on the growth of Staphylococcus aureus and Listeria monocytogenes during manufacturing,ripening and storage of Iranian white‐brined cheese

This research evaluated the effects of Mentha longifolia L. essential oil (EO) in concentrations 0, 50, 150 and 300 ppm and Lactobacillus casei (10⁸–10⁹ CFU/mL) on the growth of Staphylococcus aureus and Listeria monocytogenes during the manufacturing, ripening and storage of Iranian white‐brined cheese. The growth of the two pathogens was significantly reduced (P < 0.01) by both EO concentrations ≥50 ppm and probiotic and their combination in the standard manufacturing and storage process conditions of the cheese. Furthermore, the treatment containing 150 ppm of this EO combined with probiotic had a favourable inhibitory effect on the growth of two pathogenic micro‐organisms and also was the most appropriate treatment in sensory assessment. The synergistic effects of the above‐mentioned concentration level between the essential oil and probiotic were significant compared to other treatments, including essential oil and probiotic only. Thus, a lower concentration of this EO can be used when it is combined with this probiotic.     

Effect of technological practices on individual betalains and antioxidant activity of Columbian betalain‐rich raw materials

The effect of different technological practices on ulluco (ullucus tuberosus) and Opuntia dillenii has been scrutinised. Their stability at different pHs and temperatures of storage or processing over time was monitored. Our aim was focused on the determination of individual betalains and antioxidant activity, not previously conducted in conjunction with these raw materials. On the basis of the results, it could be asserted that the ullucus tuberosus and Opuntia dillenii were more suitable for being added to low‐acidic foods (pH 5 and 6), in the light of the higher values of betalains and antioxidant capacity (1.3‐fold higher compared with pH 4). With regard to the temperature, cold storage conditions (4 °C) were optimal to increase or maintain as possible the initial content of betalains and antioxidant capacity. After cooking (80 °C), the identified betalains completely disappeared, but the low‐acidic conditions (pH 6) favoured the greater antioxidant activity when kept at that temperature.