At semi-industrial scale it was obtained two cheeses, one with aromatic culture (CHN) and the other with also <i> L. helveticus </i> (CHN + LH). Proteolysis during ripening was evaluated using proteolytic indices, electrophoretic profile Urea-PAGE and release of free amino acids. Sensory evaluation was performed by acceptability test. It was observed a 5-fold increase in the fraction corresponding to <i> α </i><sub> s1-I </sub> -casein in the CHN cheese and 6.5 times in the CHN + LH cheese and 7.2 and 8.2 increase respectively, for total amino acids. Prato cheese with the adjunct culture showed higher acceptability in terms of overall aroma and flavor.
Prato is a typical Brazilian cheese that ranks among the most produced cheeses in the country. This type of cheese is of Danish origin, similar to Gouda and Danbo, despite its different flavor and texture [
According to Kabelová et al. [
Cheese taste and texture have strong influence depending on the types of cultures used. During cheese ripening, proteolysis of casein forms the basis for generating a wide range of peptides and amino acids, with the potential to influence cheese taste [
The CHN culture is composed of Lactococcus lactis ssp. cremoris and Lc. lactis ssp. lactis, and two cultures that promote flavor: Lc. lactis ssp. lactis biovar. diacetylactis and Leuconostoc mesenteroides ssp. cremoris. Besides lactic acid, Lc. lactis ssp. lactis biovar. diacetylactis produces acetoin and diacetyl compounds that provide aroma. Leuconostoc mesenteroides ssp. cremoris is heterofermentative, producing from glucose many compounds: lactic and acetic acids, ethanol, carbon dioxide, acetoin and diacetyl [
Lactobacillus helveticus (LH) is a thermophilic homofermentative species traditionally used as starter in the manufacture of long ripening Swiss-type cheese, Italian cheese, such as Emmental, Gruyere, Grana Padano and Parmigiano Reggiano and fermented milk beverages [
The objective of this study was to evaluate the potential of using L. helveticus B02 as an adjunct culture of mesophilic aromatic culture to improve Prato cheese ripening and sensory characteristics.
Mesophilic aromatic culture CHN-22 DVS (direct vat set) presently referred as CHN, and thermophilic culture Lactobacillus helveticus B02 DVS (LH culture) from Chr. Hansen (Valinhos, SP, Brazil) were rehydrated in 1 L of sterilized (110˚C for 10 min), reconstituted (10%, w/v) powder skimmed milk (Molico, Nestle). Prior to manufacturing the cheese, the CHN culture was activated (growth at 22˚C for 16 h) and the LH culture activated at 45˚C for 18 h.
Two batches of cheese were produced in parallel using 140 L of milk for each batch, at semi-industrial scale, using a 200 L capacity Queijomatic tank (Biasinox, Brazil). In batch 1, the CHN culture (200 mL 100 L−1) was added to the pasteurized milk. In the 2nd batch, besides the addition to the CHN culture, the LH culture was added (215 mL 100 L−1) after coagulation, according to the flowchart of Prato cheese processing (
Heat-treated milk was characterized for pH (pH meter Micronal B-375), titratable acidity (%lactic acid), and fat using the Gerber method [
After three days manufacturing and 10, 17, 24 31, 38 and 45 days of ripening the cheeses were analyzed for pH, titratable acidity and fat moisture, lactose, salt, ashes, total nitrogen, soluble nitrogen at pH 4.6 [
The pasteurized milk was analyzed for Salmonella sp. (in 25 g), Listeria mono-
cytogenes (in 25 g), coagulase-positive staphylococci (CFU g−1), total mesophilic (CFU g−1), mold and yeasts (CFU g−1), coliforms at 35˚C and 45˚C (MPN g−1) [
Cheese at 3 days following manufacture and at 10, 17, 24 31, 38 and 45 days of ripening were analyzed for proteolysis and electrophoretic profile of casein and at 3, 24 and 45 days for release of total and individual free amino acids.
Proteolysis was determined as soluble nitrogen at pH 4.6, expressed as a percentage of total nitrogen (Extension Index of Proteolysis―EIP), and as soluble nitrogen in trichloroacetic acid 12%, expressed as a percentage of total nitrogen (Depth of Proteolysis Index―DPI), determined by the macro-Kjeldahl method [
Polyacrylamide gel electrophoresis (urea-PAGE) was carried out using a vertical mini-PROTEAN® Casting System (Bio-Rad Laboratories Inc.), according to Andrews [
Free amino acids were determined by reverse phase-high performance liquid chromatography (RP-HPLC) using a Shimadzu HPLC system (Shimadzu Corporation, Japan), equipped with a Luna/Phenomenex C18 column (4.6 - 250 mm, 5 L). Identification and quantification was done by external standard (Pierce/PN 20088) with UV detection at 254 nm [
Acceptability tests were conducted at 7 and 30 days of manufacture with 100 cheese consumers, without restrictions to age, gender or social class. Three samples were presented: Prato cheese (made only with CHN starter culture), Prato cheese with adjunct culture (CHN + LH) and commercial Prato cheese (a market leader). The samples were served in a sequential monadic manner according to a complete balanced block design. The samples were evaluated according to Meilgaard et al. [
The microbiological and physicochemical results of the milk after pasteurization was in accordance with the Brazilian legislation parameters [
The composition of CHN and CHN + LH cheeses at 3 days following manufacture also complies with the legal parameters established by Brazilian regulations for moisture (36.0% - 45.9%), fat on a dry basis (45.0% - 59.9%) and pH (5.2 - 5.4) [
Microbiological characterization of CHN and CHN + LH cheeses three days following production (
Proteolysis during cheese ripening was determined by the concentration (%) of the soluble nitrogen compounds at pH 4.6-SN at pH 4.6 (extension of proteolysis) and SN 12% TCA (depth of proteolysis) related to the percentage of total nitrogen (TN), as shown in
Parameter | LD Prato Cheese | LD+LH Prato Cheese |
---|---|---|
Physico-chemical | ||
pH | 5.36a | 5.40a |
Acidity (%) | 0.6a | 0.61a |
Fat (%) | 29.39a | 29.05a |
Fat on a dry basis (%) | 53.01a | 49.12a |
Moisture (%) | 44.56a | 40.86b |
Salt (%) | 2.39a | 2.27b |
Salt humidity (%) | 5.67a | 5.04a |
Total protein (%) | 26.33a | 26.51a |
Microbiological | ||
Salmonella sp. (in 25 g) | Abs. | Abs. |
Listeria monocytogenes (in 25 g) | Abs. | Abs. |
Coagulase-positive staphylococci (CFU g−1) | <10 | <10 |
Coliforms at 30˚C - 35˚C (MPN g−1) | <3 | <3 |
Coliforms at 45˚C (MPN g−1) | <3 | <3 |
Molds and yeasts (CFU g−1) | <10 | <10 |
Abbreviations are: MPN, most probable number per milliliter; CFU g-1, colony-forming unit per milliliter; Abs., Absent.
characteristics of Prato cheese [
The electrophoretic profile (
At 45 days of ripening, the % of the relative peak area from the electrophoretograms (not shown) we observed degradation of 56% and 67% of αs1-casein fraction in the CHN and CHN + LH cheeses, respectively. The values found were very close to those found by Gorostiza et al. [
β-casein. We observed γ3-casein band fraction after 17 days of ripening, while in the cheese without the addition of an adjunct culture this fraction was evident only at 45 days of ripening. El-Soda et al. [
The individual and total free amino acid levels progressively increased in CHN and CHN +LH cheeses (P < 0.05). However, there was no significant difference in the amount of any of the free amino acids due to the use of the adjunct culture. Glutamic acid and leucine were the free amino acids found in greater quantities in the two cheeses produced. The former is known to be a main contributor to umami taste. Glutamic acid and mineral salts were the key drivers for umami taste in Cheedar cheese according to research conducted by Andersen et al. [
Amino acid | CHN Cheese | CHN + LH Cheese | ||||
---|---|---|---|---|---|---|
3 | 24 | 45 | 3 | 24 | 45 | |
Aspartic Acid. | 1.37b | 2.46b | 14.08a | 1.10b | 3.58b | 13.77a |
Glutamic acid | 7.40c | 42.53b | 197.67a | 8.43c | 55.93b | 219.76a |
Serine | 2.38c | 7.28b | 23.55a | 2.81c | 15.54b | 21.26a |
Glycine | 0.83c | 13.68b | 19.90a | 1.03c | 7.53b | 16.74a |
Histidine | 8.16b | 12.99b | 50.53a | 8.00c | 16.32b | 45.11a |
Arginine | 3.10c | 12.48b | 26.86a | 1.19c | 11.75b | 21.74a |
Threonine | 2.31c | 13.81b | 28.22a | 2.99c | 16.68b | 30.20a |
Alanine | 6.84c | 16.51b | 25.02a | 7.07c | 16.87b | 25.47a |
Proline | 20.90c | 29.90b | 41.34a | 19.22c | 27.93b | 44.88a |
Tyrosine | 10.77c | 23.63b | 40.04a | 9.58c | 22.46b | 38.94a |
Valine | 9.44c | 43.62b | 79.77a | 8.43c | 42.61b | 90.10a |
Methionine | 4.15c | 11.86b | 15.90a | 4.34c | 13.20b | 18.46a |
Cystine | 9.92ab | 9.05b | 15.20a | 10.04a | 11.02a | 21.26a |
Isoleucine | 3.89c | 15.76b | 35.67a | 7.42b | 17.55b | 39.82a |
Leucine | 15.54c | 99.92b | 205.01a | 25.22c | 103.52b | 232.72a |
Phenylalanine | 10.48c | 48.46b | 85.65a | 8.37c | 51.03b | 100.06a |
Lysine | 18.17c | 49.19b | 87.37a | 18.18c | 51.93b | 98.01a |
Tryptophan | 12.12c | 50.66b | 93.03a | 11. 78c | 68.67b | 114.66a |
Total | 149.86c | 503.55b | 1084.87a | 147.29c | 554.42b | 1215.04a |
Different letters in the same row are significantly different (Tukey’s test, P < 0.05). Abbreviations: Ac. Acid; LD: Aromatic mesophilic culture CHN-22; LH: L. helveticus B02.
According to Kabelová et al. [
Considering the sum of Glu and Asp for CHN or CHN + LH cheeses, both of them presented 19% of these umami amino acids released by ripening. However, in absolute numbers, in a 30 g portion of cheese, 63 mg and 70 mg of theses umami amino acids were found, respectively in CHN or CHN + LH samples, and that higher quantity found in the cheese made with adjunct culture could be the reason for the taste improvement observed in the sensory test.
There are few published works on the release of free amino acids in Prato cheese. In one of the published works (in which adjunct culture was not used) an 8-fold increase of free amino acids was achieved during ripening (60 days) with a final average value of 2.454 mg 100g−1 of total solids [
Moreno [
At the beginning of ripening (
Cheese flavor is one of the most important criteria for product quality and consumer acceptance [
Attribute | CHN Cheese | CHN + LH Cheese | Commercial cheese | L.S.D. |
---|---|---|---|---|
Appearance | 6.9 (1.4)ab | 7.2 (1.1)a | 6.5 (1.7)b | 0.45 |
Aroma | 6.5 (1.5)b | 7.1 (1.1)a | 6.5 (1.5)b | 0.42 |
Texture | 6.4 (1.5)a | 6.8 (1.4)a | 6.8 (1.6)a | 0.45 |
Flavor | 6.3 (1.6)b | 7.0 (1.4)a | 6.0 (1.8)b | 0.50 |
Overall Quality | 6.4 (1.6)b | 7.1 (1.3)a | 6.1 (1.8)b | 0.49 |
Results expressed as mean (standard deviation) from 100 evaluations. L.S.D. least significant difference at 5% significance level (Tukey test). For each attribute (line), values followed by the same letters are not statistically different from each other P < 0.05). Abbreviations: CHN: Aromatic mesophilic culture CHN-22; LH: L. helveticus B02.
Attribute | CHN Cheese | CHN + LH Cheese | Commercial cheese | L.S.D. |
---|---|---|---|---|
Appearance | 7.0 (1.2)b | 7.4 (0.9)a | 7.2 (1.2)ab | 0.33 |
Aroma | 6.7 (1.3)b | 7.1 (1.1)a | 6.6 (1.4)b | 0.41 |
Texture | 7.0 (1.1)a | 6.9 (1.3)a | 7.1 (1.2)a | 0.36 |
Flavor | 6.7 (1.4)ab | 7.1 (1.2)a | 6.4 (1.8)b | 0.49 |
Overall Quality | 6.7 (1.3)b | 7.3 (1.2)a | 6.5 (1.6)b | 0.43 |
Results expressed as mean (standard deviation) from 100 evaluations. L.S.D. Least significant difference at 5% significance level (Tukey test). For each attribute (line), values followed by the same letters are not statistically different from each other P < 0.05). Abbreviations: CHN: Aromatic mesophilic culture CHN-22; LH: L. helveticusB02.
At 30 days (
Therefore, the adjunct culture L. helveticus helped to improve the sensory and overall characteristics of Prato Cheese, in terms of aroma and flavor, giving it a higher quality standard when compared to cheese prepared without this culture and a commercial brand obtained at the market.
Improvements in flavor, texture and overall impression of Prato cheese with the addition of adjunct culture (L. helveticus) were observed in the work of Barros et al. [
The addition of Lactobacillus helveticus B02 as an adjunct culture of mesophilic aromatic culture CHN-22 contributed to the improvement of sensory characteristics of Prato cheese, in terms of overall aroma and flavor. The electrophoretic profile of cheeses and total free amino acids highlighted the role of L. helveticus in the degradation process of caseins, particularly β-casein.
The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Sao Paulo, Brazil) for financing the Project (12/10789-8) and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the postgraduate scholarship.
de Azambuja, N.C., Moreno, I., Gallina, D.A., Spadoti, L.M., Motta, E.M.P., Pacheco, M.T.B., de Queiroz, A.L.M. and Antunes, A.E.C. (2017) Effect of Adjunct Culture Lactobacillus hel- veticus (B02) on the Composition, Proteolysis, Free Amino Acids Release and Sensory Characteristics of Prato Cheese. Food and Nutrition Sciences, 8, 512-525. https://doi.org/10.4236/fns.2017.85035