Study of volatile compounds profiles in milk matrices using Enterococcus faecalis EstA and Rhizomucor miehei lipase.

The use of esterase/lipase enzymes of different origins in food industry is a widely employed strategy to enhance the formation of characteristic aromatic compounds derived from fat and diversify flavour. In the present work, we studied EstA enzyme of Enterococcus faecalis and a high purity Rhizomucor miehei lipase (Palatase). EstA was obtained recombinantly in Escherichia coli BL21 (DE3), and optimum esterase activity was detected at pH 6.75 and 40 °C. We evaluated the effect of the enzymes on milk mixtures prepared with different fat contents (2.8 and 6%) and structure (native or homogenized) on volatile compounds profiles. The milk fat structure before and after the application of low homogenization was characterized by dynamic light dispersion and microscopy. Native milk fat mixtures presented particles of 4.6 µm and 184 nm and homogenized mixtures had particles of 1.4 µm and 258 nm; microscopy images were in concordance with these results. Fifteen volatile compounds were identified, including ketones, esters, alcohols, and acids. We showed the key role of milk fat levels and microstructure in the nature of the volatile compounds produced by the R. miehei enzyme. Both in native or homogenized states, the highest content of fat favored a higher production of acids whereas the lowest fat level favored a higher esters production along with a more balanced volatile profile. For EstA enzyme, results showed a limited action on fat, as biosynthesis of esters only increased with the highest fat level homogenized.

Culture media based on effluent derived from soy protein concentrate production for Lacticaseibacillus paracasei 90 biomass production: statistical optimisation, mineral characterization, and metabolic activities.

The waste and by-products of the soybean industry could be an economic source of nutrients to satisfy the high nutritional demands for the cultivation of lactic acid bacteria. The aims of this work were to maximize the biomass production of Lacticaseibacillus paracasei 90 (L90) in three culture media formulated from an effluent derived from soy protein concentrate production and to assess the effects these media have on the enzymatic activity of L90, together with their influence on its fermentation profile in milk. The presence of essential minerals and fermentable carbohydrates (sucrose, raffinose, and stachyose) in the effluent was verified. L90 reached high levels of microbiological counts (∼ 9 log cfu mL-1) and dry weight (> 1 g L-1) on the three optimized media. Enzymatic activities (lactate dehydrogenase and ß-galactosidase) of L90, and its metabolism of lactose and citric acid, as well as lactic acid and pyruvic acid production in milk, were modified depending on the growth media. The ability of the L90 to produce the key flavour compounds (diacetyl and acetoin) was maintained or improved by growing in the optimized media in comparison with MRS.

Evaluation of autochthonous cultures to improve the cheese flavor: A case study in hard cheese model.

The characterization of autochthonous cultures based on their contribution to cheese flavor is an additional selection criterion for their use in cheese making. The objective of the present work was to assess the ability of three strains of mesophilic lactobacilli: Lactobacillus casei 72 (Lc72), L. paracasei 90 (Lp90), and L. plantarum 91 (Lp91), one strain of thermophilic lactobacillus: L. helveticus 209 (Lh209), and the thermophilic-mesophilic combinations, to grow and produce aroma compounds in a hard cheese model. Microbiological counts, pH, and the profiles of carbohydrates, organic acids, and volatile compounds were analyzed during incubation for 14 days at 37 ℃. The population of mesophilic lactobacilli reached levels around 8.0 log CFU ml-1 at three days, but then decreased until ∼7.0 log CFU ml-1 toward 14 days. Thermophilic lactobacillus population reached and maintained levels around 7.7 log CFU ml-1 during incubation. Carbohydrates were absent in the hard cheese model, and so no change in the pH values and in the levels of lactic acid was detected. Mesophilic lactobacilli, inoculated individually or in association with Lh209, metabolized the citric acid and produced ethanoic acid. The profiles of volatile compounds of mesophilic lactobacilli (characterized mainly by butan-2-one, 3-hydroxybutan-2-one, 3-methylbutan-1-ol, hexan-1-ol, 2-phenylethanol, and ethanoic acid) were different from the profile of thermophilic lactobacillus Lh209 (characterized mainly by heptan-2-one, ethyl acetate, isoamyl hexanoate, pentan-1-ol, decanoic acid, and 2- and 3-methylbutanal). Cooperative effects in the production of compounds related to cheese flavor, such as 3-hydroxybutan-2-one, ethyl butanoate, ethanol, pentan-2-ol, hexan-1-ol, benzeneacetaldehyde, 2-phenylethanol, and heptanoic acid, were largely evidenced between Lh209 and Lp91; in a lesser extent, cooperative effects were also found for Lh209+Lp90 for the following compounds: 3-hydroxybutan-2-one, isoamyl acetate, and ethanoic acid. Of the mesophilic lactobacilli strains evaluated, Lp91 and Lp90 would be interesting candidates for its use as adjunct cultures in hard cheeses to improve and diversify the flavor.

Influence of the culture preparation and the addition of an adjunct culture on the ripening profiles of hard cheese.

The aim of this work was to evaluate the impact of two factors on the ripening profiles of hard cooked cheeses: (F1) the growth medium for the primary and adjunct cultures, constituted by autochthonous strains: Lactobacillus helveticus 209 (Lh209) and Lactobacillus paracasei 90 (Lp90), respectively, and (F2) the addition of L. paracasei Lp90 as adjunct culture. Four types of cheeses were made: W and M cheeses in which only Lh209 was added after its growth in whey and MRS, respectively; Wa and Ma cheeses in which both strains (Lh209 and Lp90) were added after their growth in whey and MRS, respectively. Physicochemical and microbial composition, proteolysis and profiles of organic acids and volatile compounds were analyzed. According to the methodology of the cultures preparation, W and Wa cheeses showed a higher level of secondary proteolysis and lower level of primary proteolysis (P < 0·05), lower content of citric and acetic acids and higher amount of propionic acid (P < 0·05), in comparison with M and Ma cheeses. The incorporation of Lp90 increased the secondary proteolysis (P < 0·05), decreased the citric acid (P < 0·05), and increased the propionic acid only when was added after their growth in whey (P < 0·05). Both factors significantly modified the percentages of the volatile compounds grouped in chemical families; in addition, for the half of the compounds detected, significant differences were found. Based on the obtained results, the use of Lp90 as an adjunct in hard cooked cheeses, and the preincubation of the cultures in whey are strategies to accelerate the cheese ripening and to enhance the production of some characteristic compounds of this type of cheeses, such as propan-2-one, hexan-2-one, 2- and 3-methyl butanal, heptan-2-ol, acetic and 3-methylbutanoic acids and 3-hydroxy butan-2-one.

Characterization of volatile compounds produced by Lactobacillus helveticus strains in a hard cheese model.

Starter cultures of Lactobacillus helveticus used in hard cooked cheeses play an important role in flavor development. In this work, we studied the capacity of three strains of L. helveticus, two autochthonous (Lh138 and Lh209) and one commercial (LhB02), to grow and to produce volatile compounds in a hard cheese extract. Bacterial counts, pH, profiles of organic acids, carbohydrates, and volatile compounds were analyzed during incubation of extracts for 14 days at 37 ℃. Lactobacilli populations were maintained at 106 CFU ml-1 for Lh138, while decreases of approx. 2 log orders were found for LhB02 and Lh209. Both Lh209 and LhB02 slightly increased the acetic acid content whereas mild increase in lactic acid was produced by Lh138. The patterns of volatiles were dependent on the strain which reflect their distinct enzymatic machineries: LhB02 and Lh209 produced a greater diversity of compounds, while Lh138 was the least producer strain. Extracts inoculated with LhB02 and Lh 209 were characterized by ketones, esters, alcohols, aldehydes, and acids, whereas in the extracts with Lh138 the main compounds belonged to aromatic, aldehydes, and ketones groups. Therefore, Lh209 and LhB02 could represent the best cheese starters to improve and intensify the flavor, and even a starter composed by combinations of LhB02 or Lh209 with Lh138 could also be a strategy to diversify cheese flavor.

Cheese milk low homogenization enhanced early lipolysis and volatiles compounds production in hard cooked cheeses.

Homogenization applied to cheese milk has shown to increase lipolysis but its use is not spread as it can induce detrimental effects. The aim of this work was to assess the effect of low-pressure homogenization of the cream followed by pre-incubation of cheese milk on the composition, ripening index, lipolysis and volatile profiles of hard cooked cheeses. For that, control and experimental miniature Reggianito cheeses were made and analyzed during ripening (3, 45 and 90days). Homogenization had no impact on composition and proteolysis. An acceleration of the lipolysis reaction was clearly noticed in cheeses made with homogenized milk at the beginning of ripening, while both type of cheeses reached similar levels at 90days. We found the level of several compounds derived from fatty acid catabolism were noticeably influenced by the treatment applied: straight-chain aldehydes such as hexanal, heptanal and nonanal and methylketones from C5 to C9 were preferentially formed in experimental cheeses.

Spray-dried adjunct cultures of autochthonous non-starter lactic acid bacteria.

Spray-drying of lactic cultures provides direct-to-vat starters, which facilitate their commercialization and use. However, this process may alter the metabolic activity and deteriorate technological features. In this work, we assessed the influence of spray-drying on the survival and aroma production of two strains of mesophilic lactobacilli: Lactobacillus paracasei 90 and Lactobacillus plantarum 91, which have already been characterized as good adjunct cultures. The spray-drying was carried out using a laboratory scale spray and the dried cultures were monitored during the storage for the survival rate. The dried cultures were applied to two cheese models: sterile cheese extract and miniature soft cheese. The influence on the carbohydrate metabolism and the production of organic acids and volatile compounds was determined. Both strains retained high levels of viable counts in the powder after drying and during the storage at 5°C for twelve months. In addition, they also remained at high level in both cheese models during incubation or ripening. Similar profiles of carbohydrate fermentation and bioformation of volatile compounds were observed in the cheese extracts for each of the strains when tested as both fresh and dried cultures. In addition, the ability of Lb. paracasei 90 to increase the production of acetoin and diacetyl remarkably in cheese models was also confirmed for the spray-dried culture.

Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

ABSTRACT Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci.

Aminotransferases and glutamate dehydrogenase are two main types of enzymes involved in the initial steps of amino acid catabolism, which plays a key role in the cheese flavor development. In the present work, glutamate dehydrogenase and aminotransferase activities were screened in twenty one strains of lactic acid bacteria of dairy interest, either cheese-isolated or commercial starters, including fifteen mesophilic lactobacilli, four thermophilic lactobacilli, and two streptococci. The strains of Streptococcus thermophilus showed the highest glutamate dehydrogenase activity, which was significantly elevated compared with the lactobacilli. Aspartate aminotransferase prevailed in most strains tested, while the levels and specificity of other aminotransferases were highly strain- and species-dependent. The knowledge of enzymatic profiles of these starter and cheese-isolated cultures is helpful in proposing appropriate combinations of strains for improved or increased cheese flavor.

Effect of high-pressure treatment on hard cheese proteolysis.

The application of high hydrostatic pressure (HHP) treatment has been proposed to reduce the ripening time of cheese via modifications in the enzymatic activities or the substrate reactivity. Investigations on the effect of HHP on cheese proteolysis have been undertaken with either encouraging results or little effect according to the treatment conditions and the type of cheese, but information concerning the effect of HHP on the ripening of hard cooked cheese is still lacking. In this report, we describe the effect of HHP treatment on Reggianito cheese proteolysis. For that purpose, 1-d-old miniature cheeses (5.5-cm diameter and 6-cm height) were treated at 100 or 400MPa and 20°C for 5 or 10min, and control cheeses in the trial were not pressurized. All cheeses were ripened at 12°C during 90d. The HHP did not affect gross composition of the cheeses, but microbial load changed, especially because the starter culture count was significantly lower at the beginning of the ripening of the cheeses treated at 400MPa than in controls and cheeses treated at 100MPa. Cheeses treated at 400MPa for 10min had significantly higher plasmin activity than did the others; the residual coagulant activity was not affected by HHP. Proteolysis assessment showed that most severe treatments (400MPa) also resulted in cheeses with increased breakdown of αS1- and ß-CN. In addition, nitrogen content in soluble fractions was significantly higher in cheeses treated at 400MPa, as well as soluble peptides and free AA production. Peptide profiles and individual and total content of free AA in 60-d-old treated cheese were as high as in fully ripened control cheeses (90d). Holding time had an effect only on pH-4.6-soluble nitrogen fraction and plasmin activity; cheese treated for 10min showed higher values than those treated for 5min, at both levels of pressure assayed. We concluded that HHP treatments at 400MPa applied 1d after cheesemaking increased the rate of proteolysis, leading to an acceleration of the ripening process in Reggianito Argentino cheese, whereas 100-MPa treatments did not lead to significant changes.

Influence of chymosin type and curd scalding temperature on proteolysis of hard cooked cheeses.

In this work, we studied the influence of the type of coagulant enzyme and the curd scalding temperature on the proteolysis and residual coagulant and plasmin activities of a cooked cheese, Reggianito, in the interest of reducing ripening time. A two-factor experimental design was applied in two levels: type of coagulant enzyme, bovine chymosin or camel chymosin, and curd scalding temperature, 50 or 56 °C. The experimental treatments were applied in Reggianito cheese making experiments, and the samples were ripened for 90 d at 12 °C. Scalding temperature influenced residual coagulant activity; the cheeses cooked at 50 °C had significantly higher activity than those treated at 56 °C. In contrast, scalding temperature did not modify plasmin activity. Proteolysis was primarily affected by curd cooking temperature because chymosin-mediated hydrolysis of αs1 casein was slower in cheeses treated at 56 °C. Additionally, the nitrogen content in the cheese soluble fractions was consistently lower in the cheeses scalded at 56 °C than those cooked at 50 °C. A significant influence of the type of coagulant enzyme was observed, especially in the nitrogen fractions and peptide profiles, which demonstrated that camel chymosin was slightly less proteolytic; however, these differences were lower than those caused by the scalding temperature.

Technological and probiotic role of adjunct cultures of non-starter lactobacilli in soft cheeses.

The influence of two cheese-isolated Lactobacillus strains on cheese composition, acceptability and probiotic capacity was assessed. Soft cheeses with and without the addition of Lactobacillus plantarum I91 or Lactobacillus paracasei I90 were prepared. Gross composition was assessed and secondary proteolysis was described by soluble fractions and free amino acids profiles. Acceptability was determined by a panel of 98 non-trained consumers. Cheeses harboring added Lactobacillus strains were also studied in vivo to evaluate their probiotic capacity. Gross composition of the cheeses was similar for control and treated (Lactobacillus-added) cheeses. Peptidolysis increased in cheeses with added lactobacilli, which was evidenced by a higher free amino acid content. Overall, the acceptability of the cheeses was good: 65%-80% of the consumers said that they "liked very much" or "liked" the cheeses. Cheeses with L. plantarum I91 showed the highest changes in composition and proteolysis and were the most accepted ones. On the contrary, composition of cheeses with L. paracasei I90 was similar to that of the controls, but these samples were less accepted than cheeses without lactobacilli. The oral administration of cheese containing L. plantarum I91 or L. paracasei I90 proved to be safe and able to enhance the number of IgA + cells in the small intestine lamina propria of mice. The use of selected strains of NSLAB exerted a technological and probiotic role: it contributed to the standardization of cheese quality and induced benefic health effects at the gut mucosa in vivo.

Pategrás cheese as a suitable carrier for six probiotic cultures.

The viability of five single-strain and one three-strain probiotic cultures was assessed during Pategrás cheese ripening. Probiotics were inoculated into cheese-milk after a pre-incubation step - intended to improve their survival - or directly as a lyophilised culture; control cheeses without probiotics were also obtained. pH of probiotic and control cheeses was similar, except in probiotic cheeses containing the strain Lb. acidophilus B or the mixed culture. In these cases, the probiotic cheeses were more acid than their respective control cheeses. All the probiotics tested maintained counts above 107 cfu/g during the shelf-life settled for the product. Strains of the Lb. casei group: Lb. paracasei, Lb. casei and Lb. rhamnosus reached and kept the highest cell concentration during cheese ripening, followed by Lb. acidophilus and bifidobacteria. The direct addition of the probiotic cultures was more efficient than their inoculation after a pre-incubation step, for all the probiotics assayed. We have provided evidence that support the use of Pategrás cheese as a performing food-based vehicle for probiotic bacteria.

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers.

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.