Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese

This work studied the qualitative and quantitative proteolytic and lipolytic activities of Yarrowia lipolytica strains isolated from two cheese types. Randomly amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to compare the cheese strains of Y. lipolytica with strains isolated from other food products and with the type strain of the species in order to investigate the genetic diversity and occurrence of specific environmental groups. Diversity of proteolytic and especially lipolytic activity within Y. lipolytica strains isolated from dairy products was observed. In particular, the degree of specificity for saturated or unsaturated fatty acids as well as for even- or odd-numbered carbon free fatty acids (FFAs) varied among the strains. The RAPD-PCR profiles showed low genetic relatedness between many of the food isolates and the type strain of the species. Such genetic variability needs to be further evaluated. Most of the Y. lipolytica strains appeared to be specific to the particular environment from which they were isolated. However, phenotypic characteristics having technological importance in dairy products and, particularly, lipolytic activities did not correspond to the genetic differences observed by RAPD-PCR analysis.     

Variability of the lipolytic activity in Yarrowia lipolytica strains in pork fat

This work studied the variability in lipolytic activity in 35 strains of Yarrowia lipolytica inoculated in pork fat after 7 and 21 days of storage at 15 °C. The strains were able to generate three different hydrolysis profiles. In particular, the strains PO10, PO14, RO1, RO5, Y15, Y16A, Y20, B5, 7B, 7B3, 16B and 21C caused an increase with time in concentrations of C16:0, C18:0, C18:1, C18:1(Δ11) and C18:2 which were the predominant free fatty acids (FFAs). On the contrary, the strains PO1, PO19, PO23, RO22, Y12, B4, B74, GB, 5B, 5D, 27D and W29 showed an opposite trend, while the remaining ones induced no change. Because the released FFAs can be considered precursors for flavour development, the results suggest the potential use of some Y. lipolytica strains in sausage making to improve the overall aroma.     

Influence of selected factors on browning of Camembert cheese

Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+ thus leading to 16 different variants of cheese. Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body). Mn2+ promoted a significant increase of browning at both locations, whereas Yar. lipolytica had the opposite effect. Outside browning was significantly more intense when using the Pen. candidum strain with higher proteolytic activity. A significant interaction was found between Yar. lipolytica and Pen. candidum. The yeast had no effect in combination with a low proteolytic strain of Pen. candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen. candidum. We further confirmed that both strains of Pen. candidum were able to produce brown pigments from tyrosine and thus both are presumably responsible for the browning activity in this type of cheese.     

Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

Inhibition of yeasts isolated from traditional Turkish cheeses by Lactobacillus spp.

Antifungal activity of some Lactobacillus strains was determined. Yeasts were isolated and identified as Saccharomyces cerevisiae (10 of 17) and one each of Candida pseudotropicalis, C. krusei, C. lipolytica, C. lusitaniae, C. ciferrii, Torulopsis glabrata and Rhodotorula rubra . Lactobacillus strains were found to have fairly strong antifungal activity against S. cerevisiae . Of the 19 bacteria tested, L. plantarum Lp 21 seemed to have the most inhibitory effect against all of the S. cerevisiae strains except for S. cerevisiae 13. We suggest that L. plantarum Lp 21 can be used with starter cultures for cheese production.     

Controlled production of camembert-type cheeses: part III role of the ripening microflora on free fatty acid concentrations

Phenomena generating FFAs, important flavour precursors, are significant in cheese ripening. In Camembert-like cheeses, it was intended to establish the relationships between the dynamics of FFA concentrations changes and the succession of ripening microflora during ripening. Experimental Camembert-type cheeses were prepared in duplicate from pasteurised milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti, and Brevibacterium aurantiacum under aseptic conditions. For each cheese and each cheesy medium, concentrations of FFAs with odd-numbered carbons, except for 9:0 and 13:0, did not change over time. For long-chain FFAs, concentrations varied with the given cheese part (rind or core). K. lactis produced only short or medium-chain FFAs during its growth and had a minor influence on caproic, caprylic, capric, and lauric acids in comparison with G. candidum, the most lipolytic of the strains used here. It generated all short or medium-chain FFAs (4:0-12:0) during its exponential and slowdown growth periods and only long-chain ones (14:0-18:0) during its stationary phase. Pen. camemberti produced more long-chain FFAs (14:0-18:0) during its sporulation. Brev. aurantiacum did not generate any FFAs. The evidence of links between specific FFAs and the growth of a given microorganism is shown.     

Free fatty acid profiles of Reggianito Argentino cheese produced with different starters

Reggianito Argentino cheeses were made at pilot plant scale using a natural whey starter (control) and different combinations of strains of selected Lactobacillus helveticus (experimental cheeses). Free fatty acids (FFAs) were extracted from cheese samples at different ripening times, and FFAs from C_6:0 to C_18:2 were analysed by gas chromatography. The characteristics of the chromatographic profiles were studied by principal components and linear discriminant analysis were performed. Levels of individual FFAs from caproic (C_6:0) to linoleic (C_18:2) acids increased significantly $(p<0.05)$ during ripening in control and experimental cheeses. Palmitic (C_16:0) and oleic (C_18:1) acids were the most abundant FFAs throughout ripening in all cheeses. No significant differences were found between FFA profiles of control and experimental cheeses. The possibility of using selected strains of Lactobacillus to replace traditional “natural whey starter” in the production of the cheese is discussed.     

Variability of the lipolytic activity and volatile molecules production by a strain of Yarrowia lipolytica in pork fat and its dependence on environmental conditions

The effects of selected variables, i.e. temperature, water activity and yeast inoculation level, on the lipolytic pattern and volatile production by Yarrowia lipolytica Y16A (chosen on the basis of a previous screening) were assessed. The variables were varied according to a central composite design and the models obtained enabled evaluation and weighting of the effects of the independent variables on the free fatty acids (FFAs) and volatile profiles in pork fat based medium. The polynomial models showed the levels temperature, water activity of the pork fat based system and yeast strain inoculation were able to maximize the release of specific FFAs or molecules of sensory importance.     

Identification of yeasts associated with milk products using traditional and molecular techniques

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.     

Yeasts as adjunct starters in matured Cheddar cheese

Debaryomyces hansenii and Yarrowia lipolytica are typical foodborne yeast species frequently associated with dairy products and capable of predominating the yeast composition in such systems. The two species fulfil a number of criteria to be regarded as co-starters for cheesemaking. They are known for their proteolytic and lipolytic activity as well as their compatibility and stimulating action with the lactic acid starter cultures when co-inoculated. Recent studies indicated that yeasts could be included as part of starter cultures for the manufacturing of cheese, enhancing flavour development during the maturation. The potential of D. hansenii and Y. lipolytica as agents for accelerated ripening of matured Cheddar cheese has been evaluated during four cheese treatments. The interaction between the two yeast species and the lactic acid bacteria was surveyed incorporating (i) D. hansenii, (ii) Y. lipolytica, (iii) both species as adjuncts to the starter culture and (iv) a control cheese without any additions for the production of matured Cheddar cheese. The physical and chemical properties of the cheeses were monitored in order to evaluate the contribution of the yeasts to cheese maturation. The yeasts grew in association with the lactic acid bacteria without any inhibition. The yeasts species when individually added contributed to the development of bitter flavours despite accelerated development of strong Cheddar flavours. When both species were incorporated as part of the starter culture, the cheese, however, had a good strong flavour after a reduced ripening period. The cheese retained this good flavour and aroma after 9 months of production. The simultaneous application of D. hansenii and Y. lipolytica as part of the starter culture for the production of matured Cheddar cheese is proposed.     

Free fatty acid profile of Parmigiano–Reggiano cheese throughout ripening: Comparison between the inner and outer regions of the wheel

From each of twenty wheels of Parmigiano–Reggiano cheese of different ages (1–24 months), samples representative of the inner part (I, 10 cm from the round side and 7 cm from the flat side) and of the outer part (O, the rest of the cheese after removing the rind) were collected. For each sample, free fatty acids (FFAs, expressed as mg kg⁻¹ cheese fat) were determined by quantitative capillary gas chromatography. In 18- and 24-month-old cheese, total levels of FFAs (as sum of individual FFAs) were significantly (P ≤ 0.05) higher in O than I, whereas no differences between regions were observed in 1-, 6- and 12-month-old cheeses. In terms of short (C4–C8) and medium (C10–C14) chain FFAs, levels in the outer regions were higher (P ≤ 0.05) than inner regions for cheeses from 6- to 24-month-old. The content of long-chain FFAs (C16–C18:2) was higher in O than I in 18- and 24-month-old cheeses.     

The growth, properties and interactions of yeasts and bacteria associated with the maturation of Camembert and blue-veined cheeses

The growth of yeasts and bacteria were monitored during the maturation of Camembert and blue-veined cheese produced in Australia. Yeasts were prominent throughout maturation, growing to 10(5)-10(9)/g, depending on the manufacturer. Debaryomyces hansenii predominated, but there were lesser, inconsistent contributions from Yarrowia lipolytica. Of the non-lactic acid bacteria, Acinetobacter species were significant during the maturation of Camembert but not blue-veined cheeses, and grew to 10(6)-10(8) cfu/g. Staphylococcus and Micrococcus species were consistently isolated from the cheeses with Staphylococcus xylosus growing to 10(5)-10(9) cfu/g, depending on the product. Lactic acid bacteria (10(7)-10(9) cfu/g) were present throughout maturation but were not identified. Interactions between the various yeasts and bacterial isolates were examined. Several strains of D. hansenii exhibited killer activity but not against Y. lipolytica. None of the yeasts were antagonistic towards the bacteria but some strains of D. hansenii enhanced the growth of Y. lipolytica and S. xylosus. The yeast and bacterial isolates exhibited various degrees of extracellular proteolytic and lipolytic activities.     

Determination of free fatty acids in milk and dairy products by reversed-phase HPLC with fluorescence detection

A highly sensitive HPLC with fluorescence detection was developed for the determination of free fatty acids (FFAs) in milk and dairy products. For this purpose, the FFAs were extracted from small amounts of milk (1.0 mL), cheese (0.5 g) and butter (0.5 g) using Sep-Pak cartridge columns, and then derivatized to 9-anthrylmethyl esters with 9-anthryldiazomethane (ADAM). Good separations of the ADAM derivatives of 16 FFAs (C4-C18) that exist in milk, cheese and butter, which permitted quantitative estimation of their individual FFAs, were achieved by HPLC on a C18 column (Cadenza CD-C18, 150 x 3 mm i.d.) using gradient elution with methanol and water. The acid values calculated from the contents of individual FFAs were in good agreement with those obtained by the conventional titration method. These results demonstrate that the present HPLC method is simple, sensitive and precise, and could be utilized widely for determination of the FFAs in milk and dairy products.     

Effect of yeast and bacterial adjuncts on the CLA content and flavour of a washed-curd,dry-salted cheese

Three strains of Propionibacterium freudenreichii ssp. shermanii that converted free linoleic acid to conjugated linoleic acid (CLA) in laboratory media were used as adjunct strains, together with strains of Geotrichum candidum and Yarrowia lipolytica, to make a dry-salted, washed-curd cheese. Lactobacillus fermentum was included to produce ethanol (from lactose), a potential substrate for ethyl ester synthesis, while Lactobacillus rhamnosus was used to control the adventitious non-starter lactic acid bacteria population. The total (esterified plus free) level of CLA was similar in the control and experimental cheeses and remained unchanged over 4 months of ripening. Addition of linoleic acid-rich safflower oil to the cheese curd increased the concentration of free linoleic acid generated in the cheese but the CLA content did not change. Free linoleic acid was released by the yeast lipase(s) but there was no conversion to CLA. High concentrations of ethyl esters were produced in the cheeses made with added yeast, giving a fruity flavour.     

新疆塔城地区哈萨克族传统奶酪中酵母菌的分离鉴定

为保护新疆哈萨克族传统奶酪中的优良酵母菌株,从新疆塔城牧区不同牧场采集的10份哈萨克族传统奶酪样品中,分离得到44株酵母菌。采用形态学、生理生化特性鉴定、5.8S rDNA序列同源性分析相结合的方法,对分离菌株进行鉴定。共鉴定出5个种,其中34株库德毕赤酵母（Pichia kudriavzevii）,为优势菌株,6株戴尔有孢圆酵母（Torulaspora delbrueckii）,2株乳酸克鲁维酵母（Kluyve- romyces lactis ）,1株马克思克鲁维酵母（Kluyveromyces marxianus ）,1株发酵毕赤酵母（Pichia fermentans ）。结果表明,哈萨克族传统奶酪制品中所含酵母菌与其他地区的存在差异性,有其独特的酵母菌资源。     

Potential of anticlostridial Lactobacillus isolated from cheese to prevent blowing defects in semihard cheese

Five anticlostridial Lactobacillus strains isolated from cheese were selected for a mixed adjunct culture. Cheese with the mixed adjunct culture (experimental) and without (control) was made in triplicate and ripened as vacuum‐packed and surface‐ripened cheese. Cheese gross composition was similar. Excessive gas formation occurred only in control cheeses. In contrast to control cheeses, the experimental cheeses were dominated by the added adjunct Lactobacillus strains (repetitive‐PCR). Casein breakdown was not influenced, however, the total amount of amino acids and pH was slightly lower in the experimental cheeses. Anticlostridial nonstarter Lactobacillus strains have potential as protective adjunct cultures against blowing defects in cheese.     

Effect of a bacteriocin-producing Lactococcus lactis strain and high-pressure treatment on the esterase activity and free fatty acids in Hispánico cheese

The effect of milk inoculation with a bacteriocin-producing (BP) culture and of high-pressure (HP) treatment of 15-day-old Hispánico cheeses (400 MPa, 5 min, 10 °C), separately or combined, on the release of intracellular esterases and cheese lipolysis was investigated. Esterase activity and free fatty acids (FFAs) content increased during ripening of Hispánico cheese and palmitic, oleic and stearic acids being the most abundant FFAs. On day 15, the highest esterase activity was recorded for HP-treated BP cheese. The activity for HP-untreated BP cheese was the next highest. No difference in the activities was found between HP-treated and untreated cheeses made without BP culture. Total FFAs on day 15 were at a lower concentration in BP cheeses than in cheeses made without BP culture, probably due to the lower pH values of the former. The rate of total FFA accumulation from day 15 to day 50 was higher in BP cheeses (31.1–32.1% increase) than in cheeses made without BP culture (19.3–21.7% increase). The highest total FFA concentration on day 50 (612 mg kg⁻¹) was found for HP-untreated cheese made without BP culture.     

Contribution of autochthonous non-starter lactobacilli to proteolysis in Caciocavallo Pugliese cheese

Caciocavallo Pugliese cheese was manufactured according to a traditional protocol and with added autochthonous Lactobacillus paracasei subsp. paracasei and L. parabuchneri strains, isolated from a well-flavoured Caciocavallo Pugliese cheese, to evaluate their contribution to the cheese biochemical characteristics. Using a “two step REP-PCR” protocol, L. paracasei subsp. paracasei strains were shown to sustain high viability during ripening, while the L. parabuchneri strains were not recovered. The inoculated cheese showed higher levels of free amino acids, as well as differences in the profiles of individual free amino acids in comparison with the control cheese. The addition of autochthonous Lactobacillus strains with interesting technological properties in Caciocavallo Pugliese cheese manufacturing could make it feasible to improve cheese processing, while still maintaining the sensory characteristics of this typical pasta-filata cheese.     

High-Pressure Processing for the Control of Lipolysis,Volatile Compounds and Off-odours in Raw Milk Cheese

Build-up of flavour compounds throughout ripening of raw milk cheeses may result in strong over-ripening notes during refrigerated storage. In order to control the formation of free fatty acids (FFAs) and volatile compounds, and the appearance of off-odours, raw milk cheeses were high-pressure-processed (HPP) 21 or 35 days after manufacture at 400 or 600 MPa. Ripening proceeded at 8 °C until day 60 and, afterwards, cheeses were held at 4 °C until day 240. The effect of HPP on the formation of FFAs and volatile compounds was dependent on pressure level and cheese age at the time of treatment. On day 60, acetic and propionic acids, branched-chain FFAs and short-chain FFAs showed the lowest (p?<?0.05) concentrations in cheeses treated at 400 or 600 MPa on day 21, while medium- and long-chain FFAs were at similar levels in all cheeses. HPP influenced significantly (p?<?0.05) 84 out of the 94 volatile compounds found in cheese. On day 60, the lowest (p?<?0.05) concentrations of acids, alcohols and esters were recorded for cheeses treated at 400 or 600 MPa on day 21, and the lowest (p?<?0.05) concentrations of ketones for cheeses treated at 400 MPa on days 21 or 35. On day 240, all HPP cheeses showed lower (p?<?0.05) concentrations of aldehydes, esters and, particularly, sulphur compounds than control cheese, which exhibited putrid and rancid off-odours from day 120 onwards. Principal component analysis combining FFAs and volatile compounds discriminated 240-day control cheese from 120-day control cheese and both from the rest of cheeses.