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Starter bacteria as producers of CLA in ripened cheese

PAULA M. OLIVO GERALDO TADEU DOS SANTOS BRUNA M. RODRIGUES MILENE P. OSMARI FRANCILAINE ELOISE DE MARCHI GRASIELE S. MADRONA BRUNA C. AGOSTINHO MAGALI S.S. POZZA About the authors

Abstract

The profile of polyunsaturated fatty acids in cheeses obtained through fermentation by lactic acid bacteria Lactobacillus helveticus and Streptococcus thermophilus were evaluated. The milk used to make the cheeses came from cows fed with flaxseed oil and annato. The cheeses presented microbiological and physic-chemical quality with in the standards established by the legislation for Staphylococci and Listeria. With maturation, there was a reduction in the coliform values ​​for both treatments. Regarding the counts of lactic acid bacteria, these remained viable until the 30th day of maturation and the proteolytic bacteria decreased. For antioxidant capacity, the treatment containing the combination of the strains obtained high ABTS values. There was no significant difference between the treatments with respect to the color of the samples. For texture, there was a significant difference for the parameters cohesion and elasticity. No increase in CLA content was observed in the form of its two main isomers, however, the levels of polyunsaturated fatty acids were increased

Key words
Annatto; fatty acid profile; flaxseed oil; lactic acid bacteria; microbiology

INTRODUCTION

Currently, there is a tendency to relate health problems to a diet of inferior quality. The association between the consumption of dairy products, mainly cheeses, and health is a factor that is considered in the production and trade of these foods (Terpou et al. 2017TERPOU A, GIALLELI AI, BOSNEA L, KANELLAKI M, KOUTINAS AA & CASTRO GR. 2017. Novel cheese production by incorporation of sea buckthorn berries (Hippophaerhamnoides L.) supported probiotic cells. LWT 79: 616-624.) because it determines the consumption of these products.

Cheeses are associated with high levels of saturated long chain fatty acids, which are considered harmful if consumed in high quantities in diets because they cause several metabolic disorders and diseases. However, with the applications of different production technologies, such as varying the composition of milk, breed of animals, stage of lactation and diet of the animals, this obstacle has been reduced. Due to the great diversity of dairy products, enriched or not with unsaturated fatty acids, there have been some improvements in the final product, making dairy products healthier and potentially beneficial (González-Martín et al. 2017GONZÁLEZ-MARTÍN MI, PALACIOS VV, REVILLA I, VIVAR-QUINTANA AM & HERNÁNDEZ-HIERRO JM. 2017. Discrimination between cheeses made from cow’s, ewe’s and goat’s milk from unsaturated fatty acids and use of the canonical biplot method. J Food Compos Analysis 56: 34-40.).

The composition of fatty acids in foods is of great importance, especially polyunsaturated fatty acids, such as omega-3 and omega-6 families, which are attributed numerous benefits to humans (Perini et al. 2010PERINI JADL ET AL. 2010. Ácidos graxos poli-insaturados n-3 e n-6: metabolismo em mamíferos e resposta imune. Rev Nut 23(6): 1075-1086.). Conjugated linoleic acid (CLA) is a group of positional and geometric isomers of linoleic acid that are naturally synthesized in the rumen of animals and mainly derived from milk-derived foods and meat from ruminants (Andrade et al. 2012ANDRADE JC ET AL. 2012. Production of conjugated linoleic acid by food-grade bacteria: A review. Int J Dairy Technol 65(4): 467-481.).

The strategies to increase the amount of CLA in milk may involve the addition of dietary sources to animal feed (Jones et al. 2005JONES EL ET AL. 2005. Chemical, physical, and sensory properties of dairy products enriched with conjugated linoleic acid. J Dairy Sci 88(8): 2923- 2937.) or by synthesis by some strains of bacteria such as bifidobacteria and lactic acid bacteria with linolenic acid as a starting material (Gorissen et al. 2010GORISSEN L, RAES K, WECKX S, DANNENBERGER D, LEROY F, DE VUYST L & DE SMET S. 2010. Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species. Appl Microbiol Biotechnol 87(6): 2257-2266.). Since cheese processing also involves bacterial fermentation, some studies have been conducted to observe the synthesis of CLA by bacteria in this type of product, as well as the effect of process steps that maintain the CLA content (Lucatto et al. 2014LUCATTO JN, DE BRANDÃO SN & DRUNKLER DA. 2014. Ácido linoleico conjugado: estrutura química, efeitos sobre a saúde humana e análise em lácteos. Rev Inst Laticínios Cândido Tostes 69(3): 199-211.).

In addition, the consumption of dairy products containing probiotic bacteria, such as Lactobacillus and Bifidobacterium, also adds value to the commercial product and assists in the maintenance of the intestinal microbiota, guaranteeing health and greater well-being through its consumption.

The objective of this study was to evaluate the physicochemical and microbiological characteristics and the fatty acid profile of ripened cheese produced with milk enriched with a polyunsaturated fat source and fermentation promoted by the addition of two commercial cultures containing Lactobacillus helveticus and Streptococcus thermophilus.

MATERIALS AND METHODS

The experiment was carried out at the Experimental Farm of Iguatemi (FEI), belonging to the Univesidade Estadual de Maringá (UEM), Maringá/Paraná/BR. The experimental protocol was approved by the Committee on Ethics in Animal Use in Experimentation of the Universidade Estadual de Maringá, PR (No. 6450240117).

The milk used for the study was from four Holstein cows within ± 120 days of lactation. The cows received a diet consisting of roughage concentrate, which included annatto seeds (1.5% DM) and flaxseed oil (3% DM). For cheese production, milk was collected for five days in four periods of animal feeding (16 feeding days and 5 days of milk collection, totaling 21 days), totaling 96 cheese units.

For the chemical analyses of the milk (Table I), the samples were conditioned in a plastic bottle containing Bronopol® (2-bromo-2-nitropropane-1,3-diol), preservative and analysed in an Ekomilk Total (Cap-Lab, São Paulo, Brazil).

Table I
Physico-chemical composition of the milk used in the production of ripened cheeses.

To quantify the milk fatty acids, the methodologies proposed by Murphy et al. (1995)MURPHY JJ, CONNOLLY JF & MCNEILL GP. 1995. Effects on milk fat composition and cow performance of feeding concentrates containing full fat rapeseed and maize distillers’ grains on grass-silage based diets. Livest Prod Sci 44(1): 1-11. and method 5509 of ISO (1978) were used using an Agilent gas chromatograph, model 7890a (Table II).

Table II
Profile of fatty acids from milk used in ripened cheese production.

Dairy cultures “SH type”, Lyofast SH 092 F, SACCO®, containing Streptococcus thermophilus (S. thermophilus), Lactobacillus helveticus (L. helveticus), Lyofast LH 091, and SACCO® (L. helveticus) were used to manufacture the cheeses (05 UC/100 litres of milk).

For the elaboration of the cheeses, 48 litres of pasteurized milk (65°C/30 min), calcium chloride w/v (50 g for 100 litres of milk), milk cultures LH or SH and liquid coagulant v/v (Aspergillus niger 7 ml/10 L - HA-LA®-CHR, Denmark) were used. After cutting, the cheese cloth was heated to 45 °C and then molded (JandaPlast, model RH-1000). The curd cooking temperature can reach 55 to 60 °C for very hard cheeses (Ordóñez 2005). The cooking temperature used in this study was chosen because it was the optimal temperature for the activation of S. thermophilus. After 12 hours, the cheeses were salted with 2% salt (w/w) and kept in a BOD-type oven for 30 days/12°C with a relative humidity of ± 60.5%.

The water activity (Aqualab® 4TE, Decagon, São Paulo, Brazil), pH (digital pH metre, Tecnal Tec-5), titratable acidity (Lutz 2008), color (Konica Minolta), dry matter, mineral matter, crude protein (AOAC 1992AOAC. 1992. AOAC Official Methods of Analysis. Association of Official Agricultural Chemists. Washington, D.C., 15th 1: 136-138.) and fat content (Bligh & Dyer 1959BLIGH EG & DYER WJ. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Phys 37(8): 911-917.) were determined in the cheese samples at 0, 10, 20 and 30 days of maturation.

The instrumental color was determined using a Konica Minolta chromometer (Konica Minolta, Model CR 400/410, Japan), using the CIELAB system (CIE 1996CIE - Commission Internationale de l’Éclairage. 1996.Colourimetry. Vienna: CIE publication, 2nd ed.). Measurements were performed in triplicate with the previously calibrated apparatus, using pieces from the inside and outside of the cheese.

The samples were diluted in peptone water to perform the microbiological analyses (AOAC 1992AOAC. 1992. AOAC Official Methods of Analysis. Association of Official Agricultural Chemists. Washington, D.C., 15th 1: 136-138.), and the lactic acid bacteria (MRS agar- De Man, Rogosa e Sharpe, Himedia) were evaluated. Proteolytic mesophilic bacteria were grown on PCA-Himedia agar plus 1 % reconstituted skim milk (10 %) and coliforms were detected with VRB agar (Violet Red Bille agar, Himedia). At 30 days, the presence of Listeria monocytogenes and Staphylococcus aureus were evaluated (AOAC 1992AOAC. 1992. AOAC Official Methods of Analysis. Association of Official Agricultural Chemists. Washington, D.C., 15th 1: 136-138.). Samples were incubated at 35 °C for 48 hours.

To determine the lipid profile of cheeses, lipids were extracted from the samples according to Bligh & Dyer (1959)BLIGH EG & DYER WJ. 1959. A rapid method of total lipid extraction and purification. Can J Biochem Phys 37(8): 911-917.. Afterwards, the lipids were esterified according to ISO (1978) method 5509 for analysis using an Agilent gas chromatograph (Trace GC 52 Ultra, Thermo Scientific, West Palm Beach, Florida, USA) self-sampler equipped with a flame ionization detector at 250 °C and a fused silica capillary column (100 m long, 0.25 mm internal diameter and 0.20 μm, Restek 2560). Fatty acid quantification of the sample was performed by comparing the retention time of fatty acid methyl esters to standard samples (Sigma Aldrich).

The antioxidant compounds were evaluated in the cheese samples after 0, 10, 20 and 30 days of storage with the radical sequestration method involving 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as described by Re et al. (1999)RE R, PELLEGRINI N, PROTEGGENTE A, PANNALA A, YANG M & RICE- EVANS C. 1999. Antioxidant activity applying in improved ABTS radical cation de colourization assay. Free Radic Biol Med 26(9-10): 1231-1237. expressed in ET μM (Zhu et al. 2002ZHU QY, HACKMAN RM, ENSUNSA JL, HOLT RR & KEEN CL. 2002. Antioxidative activities of oolong tea. J Agric Food Chem 50(23): 6929-6934.).

For the analysis of the texture profile (TPA), the Brookfield texture analyzer-CT III (Engineering Laboratories, INC., Middleboro, MA, USA) was used in the following configurations: TPA; test speed, 1 mm/s; compression distance, 5 mm; and a TA4 cylindrical acrylic probe of 38 mm. The variables measured for TPA were hardness, chewing, cohesiveness and elasticity.

The data were analysed by ProcMixed of SAS 9.3 (2013)SAS Institute. 2013. StatisticalAnalysis System: userguide [CD-ROM]. Version 9.3. Cary (NC): SAS Insitute Inc., which tested the interaction between treatment and time, and a linear and quadratic contrast study was performed for storage times.

RESULTS

For the evaluated times, the dry matter (DM) contents increased as a function of maturation day, with a consequent decrease in moisture values (P<0.001). The values of dry matter were significant for time (P<0.001) and for the treatment x time interaction (P<0.0261), presenting linear contrast (P<0.0001) (Table III).

Table III
Bromatological composition of ripened cheeses.

With regard to moisture, there was a linear effect, and the levels were reduced during maturation. For ash content, there was a significant effect for treatment (P=0.0312) and for time (<0001) but not for the treatment x time interaction (P<0.6919), presenting a linear behaviour (P=0. 0033).

The values for ethereal extract increased with time P<0.0001 because with the decrease in moisture and the increase in the dry matter, there was an increase in the concentration of the cheese components, thus linearly increasing the amount of EE present.

There was no significant difference between the treatments and the time x treatment interaction (P>0.10) for the pH and Aw values; however, the pH and Aw values had significant differences for time. The values of titratable acidity and pH were not congruent, and acidity values were significant for the time and the treatment x time interaction (Table IV).

Table IV
Physico-chemical composition of ripened cheese.

High coliforms counts were found (Table V). There were significant differences for the treatments (P=0.0352) and for maturation times (P<0.0001), with a reduction in counts during maturation. For lactic acid bacteria counts, significant differences were observed for the maturation times (P=0.0107) and for the treatment x time interaction (P=0.0212), which exhibited quadratic behaviour. The microbiological count of proteolytic bacteria showed no significant difference for the treatments (P>0.05) but presented significant differences for time, decreasing throughout storage. Listeria and Staphylococcus aureus were not present at 30 days of maturation.

Table V
Parameters of microbiological quality of ripened cheese.

For color (Table VI), there was no difference for the parameters a* and b*, and no color change was observed as a function of cheese fermentation by Streptococcus or L. helveticus. For the treatment x time interaction, the color parameter L* was significant (P=0.0096).

Table VI
Instrumental colors of ripened cheese.

The values obtained for the texture were significant for treatment, time and the treatment x time interaction, only for cohesiveness and elasticity.

For the chewable characteristic, the values were significant for the treatment and time, with an increase in values for the storage time (linear effect P<0.001) (Table VII).

Table VII
Antioxidant capacity of the ABTS radical (ET μM) and texture parameters of ripened cheese.

The values found for the antioxidant capacity of the ABTS radical in the equivalent Trolox (ETμM) showed that there were significant differences for the treatment and for the treatment x time interaction (P<0.001), the highest value was for the Lh + St treatment (669.5 ETμM).

Fatty acids (Table VIII), such as C20:3 n6; C20:3 n3; C22:1 n9; C20:4 n6; C23:0; C22:2; C24:0; C20:5 n3; C24:1 and C22:6 n3, were found sporadically but were used to calculate the values of mono-unsaturated, saturated and polyunsaturated fatty acids.

Table VIII
Profile of fatty acids in samples of ripened cheese (mg/g fat).

There was a significant difference in the fatty acid profiles between the treatments (P<0.05), with the treatment containing S. thermophilus + L. helveticus having the highest levels of C18:0 (stearic acid) at 30 days of maturation and C19:1 n9 (oleic acid) at 10 days of maturation. The n3:n6 ratio was significant (P<0.05) for treatment, with the highest values observed in the Lh + St treatment cheese.

The highest levels of C18:3 n3 (alpha-linolenic acid) 1.2187 mg/g at the 20 days of maturation, C18:2 n6 t (linoleic acid) 2.0335 mg/g at the 20 days of maturation, fatty acid ratio polyunsaturated 0.1023 mg/g at the 20 days of maturation were observed for the treatment containing only L. helveticus. The saturated fatty acids presented a lower content in the cheese produced with L. helveticus, with an average of 30.6662 mg/g in relation to the cheese produced with the other bacteria, with a value of 64.8814 mg/g.

In the present study, the cheese produced with L. helveticus showed no increase in the CLA (conjugated linoleic acid) content in the form of its two major isomers: cis-9, trans 11 and trans 10, cis 12. However, the treatment containing only L. helveticus exhibited a higher concentration of C18:3 n3 (alpha-linolenic), presenting a higher value at 20 days of maturation (1.2187 mg/g).

The milk presented an average of 0.2387 mg/g C18:3 n3 (alpha-linolenic), and the cheese produced with Lh had an average concentration of 0.8138 mg/g. This concentration was four times higher, indicating the production of this fatty acid by metabolic routes during fermentation, which can improve the characteristics of the final product. These results appear to indicate that during ripening, CLA can be formed from linoleic acid through the action of the primary or secondary cultures besides the fact that

the continuous release of free linoleic acid during cheese ripening and, as a consequence, the possible formation of yet additional CLA (Sieber et al. 2004SIEBER R, COLLOMB M, AESCHILIMANN A, JELEN P & EYER H. 2004. Impact of microbial cultures on conjugated linoleic acid in dairy products –a review. Int Dairy J, p. 1-15.).

DISCUSSION

Evaluated cheeses are classified as medium and low humidity (Brasil 1996BRASIL. 1996. Portaria n. 146, de 07 de março de 1996. Regulamentos Técnicos de Identidade e Qualidade dos Produtos Lácteos. Ministério da Agricultura e do Abastecimento. Secretaria Nacional de Inspeção de Produtos de Origem Animal. Diário Oficial da União, Brasília, 11 mar.). According to Salazar-Montoya et al. (2018)SALAZAR-MONTOYA JA, GONZÁLEZ-CUELLO R, FLORES GIRÓN E & RAMOS-RAMÍREZ EG. 2018. Effect of free and microencapsulated Lactococcus lactis on composition and rheological properties of Manchego-type cheeses during ripening. Food Res Int 105: 59-64., by evaluating the use of Lactococcus lactis during the maturation of Manchego cheese, a gradual decrease in moisture emerged after 15 days of maturation as a result of the syneresis provided by the rearrangement of the protein network, resulting in large amounts of whey expulsion, a behaviour similar to that observed in these experiments. The moisture of the cheese is responsible for directly influencing its consistency, and during maturation, the intensity of dehydration depends on the size and shape of the cheese, as well as on the environmental conditions in which the maturation takes place (Beresford et al. 2001BERESFORD TP, FITZSIMONS NA, BRENNAN NL & COGAN TM. 2001. Recent advances in cheese microbiology. Int Dairy J 11(4-7): 259-274.).

The ash contents were in agreement with the literature recommended for ripened cheese, which ranges from 1.0% to 6.0% (Gomes 1997GOMES JC. 1997. Análise de Alimentos. Viçosa: Departamento de Tecnologia de Alimentos/UFV, p. 158.). Ferreira & Freitas Filho (2008)FERREIRA WL & DE FREITAS FILHO JR. 2008. Avaliação da Qualidade Físico-química do Queijo Coalho Comercializado no Município de Barreiros-PE. Rev Bras Tecnol Agroind 2(1). and Uliana & Rosa (2010)ULIANA GC & ROSA CS. 2010. Avaliação físico-química e sensorial de queijos coloniais com adição de extrato hidrossolúvel de soja e farelo de soja. Alim Nutr Araraquara 20(3): 485-490. studied artisanal cheeses (colonial and rennet) and obtained ash contents from 3.85% to 4.31% and 2.77% to 2.87%, respectively.

The oscillation in protein values may have occurred because of microbial activity and casein degradation may have been the result of rennet enzymes (Hynes et al. 2003HYNES ER, BERGAMINI CV, SUÁREZ VB & ZALAZAR CA. 2003. Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus. J Dairy Sci 86(12): 3831-3840.). L. helveticus has proteases attached to the cell envelope and intracellular peptidases that can be released into the cheese matrix during autolysis. Consequently, L. helveticus has also been used to accelerate protein degradation and improve flavour development during cheese maturation (Moser et al. 2017MOSER A, BERTHOUD H, EUGSTER E, MEILE L & IRMLER S. 2017. Detection and enumeration of Lactobacillus helveticus in dairy products. Int Dairy J 68: 52-59.).

The variation in pH may be dependent on the buffering capacity of the cheese and due to the amount of proteins and minerals (Narimatsu et al. 2003NARIMATSU A, DORNELLAS JRF, SPADOTI LM, PIZAIA PD & ROIG SM. 2003. Avaliação da proteólise e do derretimento do queijo prato obtido por ultrafiltração. Ciênc Tecnol Aliment 23: 177-182.) (Table IV). The process of acidification continued during ripening, which, except for the high production of lactic acid, is related to the low buffering capacity of the mass of the cheese. This is a consequence of the demineralization undergone during coagulation and the removal of the whey (Havranek et al. 2014HAVRANEK J, KALIT S, ANTUNAC D & SAMARŽIJA D. 2014. Cheese making (in Croatian). Zagreb: Croatian. Dairy Union.).

In relation to the increasing levels of acidity, these can be justified by the possible action of lactic acid bacteria (Faion et al. 2015FAION AM ET AL. 2015. Influence of the addition of natural antioxidant from mate leaves (Ilex paraguariensis St. Hill) on the chemical, microbiological and sensory characteristics of different formulations of Prato cheese. J Food Sci Technol 52(3): 1516-1524.). Artisanal cheeses are usually hand-pressed, thus presenting whey retention, interfering with the amount of lactose eliminated in the serum (Alinovi et al. 2018ALINOVI M ET AL. 2018. Effect of fermentation-produced camel chymosin on quality of Crescenza cheese. Int Dairy J 84: 72-78.). In addition, there is less elimination of lactose and higher amounts of substrates for fermentation; therefore, a greater amount of lactic acid can be produced, which may justify the lack of standardization presented for acidity and, consequently, influence the percentages of these parameters.

Another preponderant factor in the physical-chemical composition of cheeses is the Aw value, which presented a rapid decrease that can be justified as a function of water loss by evaporation and the hydrolysis of proteins to peptides and amino acids and of triglycerides to glycerol and fatty acids (Beresford et al. 2001BERESFORD TP, FITZSIMONS NA, BRENNAN NL & COGAN TM. 2001. Recent advances in cheese microbiology. Int Dairy J 11(4-7): 259-274.).

Lactic acid bacteria (LAB) produce lactic acid, which accelerates the coagulation of milk, aiding in serum syneresis and contributes to taste, form and texture (Awad et al. 2007AWAD S, AHMED N & EL SODA M. 2007. Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development. Food Chem 104(3): 1192-1199.). During cheese manufacturing and maturation, the composition of the lactic microbiota undergoes several changes, according to changes in environmental conditions, such as increased lactic acid and decreased pH (Di Cagno et al. 2006DI CAGNO R, DE ANGELIS M, LIMITONE A, FOX PF & GOBBETTI M. 2006. Response of Lactobacillus helveticus PR4 to heat stress during propagation in cheese whey with a gradient of decreasing temperatures. Appl Environ Microbiol 72(7): 4503-4514.).

Delamare et al. (2012)DELAMARE APL, DE ANDRADE CCP, MANDELLI F, DE ALMEIDA RC & ECHEVERRIGARAY S. 2012. Microbiological, physico-Chemical and sensorial characteristics of serrano, an artisanal brazilian cheese. Food Nut Sci 3(08): 1068., evaluated Serrano cheese for mesophilic bacteria, a group to which lactic acid bacteria belong, and stated that there may be a variation of 4.0 to 9.0 log CFU/g in cheeses produced with unpasteurized milk. Paiva et al. (2015)PAIVA VN, CUNHA ALFS, DA CRUZ RR, SOBRAL D, DE SOUZA R M & PINTO MS. 2015. Efeito da adição do fermento natural sobre a contagem de bactérias láticas em queijo minas artesanal do serro. Rev Inst Laticínios Cândido Tostes 70(5): 279-285. evaluated the natural starter addition of Minas artisanal cheese from Serro for over 60 days of maturation and noticed a decrease in LAB during the storage period of 8.20 log CFU/g to 7.90 log after 30 days in storage. Gursoy et al. (2018)GURSOY O, KÜÇÜKÇETİN A, GÖKÇE O, ERGİN F & KOCATÜRK K. 2018. Physicochemistry, microbiology, fatty acids composition and volatile profile of traditional Söğle tulum (goat’s skin bag) cheese. An Acad Bras Cienc 90: 3661-3674. evaluated cheeses produced from goat and sheep milk and obtained pH values of approximately 5.2 and values of lactic acid bacteria of 7.84 log CFU/g at 3 months of maturation.

The proteolytic microorganisms did not present significant differences between the evaluated treatments (P>0.05) since both treatments contained L. helveticus, a bacterium belonging to this microbial group. Proteolysis is an important factor to be considered during cheese maturation.

Although typically proteolytic microorganisms are undesirable, certain lactic acid bacteria have proteolytic activity, which is very important during maturation (Perry 2004PERRY KS. 2004. Queijos: aspectos químicos, bioquímicos e microbiológicos. Quím Nova 27(2): 293-300.). L. helveticus has biochemical pathways responsible for producing flavour compounds. Proteolysis is a pre-requisite for the growth of lactic acid bacteria and the subsequent degradation of milk proteins (casein), leading to the release of peptides and free amino acids (Forsythe 2002FORSYTHE SJ. 2002. Microbiologia da Segurança Alimentar. A Flora Microbiana dos Alimentos: Alimentos fermentados Tondo EC Ed. Artmed Editora, p. 132-147., Moulay et al. 2006MOULAY M, AGGAD H, BENMECHERNENE Z, GUESSAS B, HENNI D & KIHAL M. 2006. Cultivable lactic acid bacteria isolated from Algerian raw goat’s milk and their proteolytic activity. World J Dairy Food Sci 1(1): 12-18. ISSN 1817-308X.). This effect is of great importance for the development of flavour, aroma and texture of the finished product (Forsythe 2002FORSYTHE SJ. 2002. Microbiologia da Segurança Alimentar. A Flora Microbiana dos Alimentos: Alimentos fermentados Tondo EC Ed. Artmed Editora, p. 132-147.) if the bacteria do not produce bitter peptides.

Coliforms are considered important parameters to evaluate the microbiological quality of cheeses and are affected mainly by the decrease in water activity, increase in acidity due the lactic acid bacteria and secondary microbiota (Alexandre et al. 2002ALEXANDRE DP, SILVA MR, SOUZA MR & SANTOS WLM. 2002. Antimicrobial activity of lactic acid bacteria from artisanal minas cheese against indicator microorganisms. Arq Bras Med Vet Zootec 54(4): 424-428., Manolopoulou et al. 2003MANOLOPOULOU E, SARANTINOPOULOS P, ZOIDOU E, AKTYPIS A, MOSCHOPOULOU E, KANDARAKIS IG & ANIFANTAKIS EM. 2003. Evolution of microbial populations during traditional Feta cheese manufacture and ripening. Int Food Microbiol 82(2): 153-161. and Caridi et al. 2003CARIDI A, MICARI P, CAPARRA P, CUFARI A & SARULLO V. 2003. Ripening and seasonal changes in microbial groups and in physico-chemical properties of the ewes’ cheese Pecorino del Poro. Int Dairy J 13(2-3): 191-200.). The coliforms found in the product indicate contamination after pasteurization (Birollo et al. 2001BIROLLO GA, REINHEIMER JA & VINDEROLA CG. 2001. Enterococcivs non-lactic acid microflora as hygiene indicators for sweetened yoghurt. Int J Food Microbiol 18: 1-8.), which may have occurred in the present study because although pasteurized milk was used, recontamination may have occurred.

The use of annatto and lactic acid bacteria was not effective in the decimal reduction of this microbial group (Table V), although studies conducted by Gonçalves et al. (2005)GONÇALVES AL, FILHO AA & MENEZES H. 2005. Estudo comparativo da atividade antimicrobiana de algumas árvores nativas. Arq Inst Biol 72(3): 353-358. confirmed the antimicrobial action of annatto “in vitro”. In addition, the bioprotective effect of LAB on cheese was verified by authors such as Favaro et al. (2015)FAVARO L, PENNA ALB & TODOROV SD. 2015. Bacteriocinogenic LAB from cheeses–application in biopreservation? Trends Food Sci Technol 41(1): 37-48. and (Montel et al. 2014MONTEL MC, BUCHIN S, MALLET A, DELBES-PAUS C, VUITTON DA, DESMASURES N & Berthier F. 2014. Traditional cheeses: rich and diverse microbiota with associated benefits. Int J Food Microbiol 177: 136-154.) and Al-Gamal (2019)AL-GAMAL MS, IBRAHIM GA, SHARAF OM, RADWAN AA, DABIZA NM, YOUSSEF AM & EL-SSAYAD MF. 2019. The protective potential of selected lactic acid bacteria against the most common contaminants in various types of cheese in Egypt. Heliyon 5(3): e01362., who found a reduction of up to 95% in food pathogens such as Escherichia coli, Staphylococcus, Salmonella, Pseudomonas aeruginosa and Bacillus cereus by the L. helveticus strain CNRZ32. The cheeses color (Table VI) may be related to different internal and external factors, and the opacity of the cheeses (L*) can be affected by the degree of matrix hydration.

The variations may be due mainly to the initial composition of milk and whey and the cheese making technology (Pena-Serna et al. 2016PENA-SERNA C, PENNA ALB & LOPES JF. 2016. Zein-based blend coatings: Impact on the quality of a model cheese of short ripening period. J Food Eng 171: 208-213.). According to Ginzinger et al. (1999)GINZINGER W, JAROS D, LAVANCHY P & ROHM H. 1999. Raw milk flora affects composition and quality of Berg Kase. 3. Physical and sensory properties, and conclusions. Lait 79(4): 411-421., the color parameter b* is strongly correlated with the yellowish color and may be linked with the maturation time. In this study annato did not influence b*. Buffa et al. (2001)BUFFA MN, TRUJILLO AJ, PAVIA M & GUAMIS B. 2001. Changes in textural, microstructural, and colour characteristics during ripening of cheeses made from raw, pasteurized or high-pressure-treated goats’ milk. Int Dairy J 11(11-12): 927-934. analysed the color change in cheeses with and without the pasteurization process during the 60-day maturation and found that the a* value remained constant up to 30 days (0.55), the L-value decreased (91.53) and the value b* increased (8.51).

The elasticity is a measure of the recovery of the original, undeformed condition after the first compressive force is removed and the cohesiveness is the amount a cheese can be deformed before breaking (Ong et al. 2012ONG L, DAGASTINE RR, KENTISH SE & GRAS SL. 2012. The effect of pH atrenneting on the microstructure, composition and texture of Cheddar cheese. Food Res Int 48(1): 119-130.). These attributes are affected by milk composition, cheese production and procedures, microorganisms, maturation and moisture, pH and soluble calcium (Lucey et al. 2003LUCEY JA, JOHNSON ME & HORNE DS. 2003. Invited review: perspectives on the basis of the rheology and texture properties of cheese. J Dairy Sci 86(9): 2725-2743., McMahon et al. 2005MCMAHON DJ, PAULSON B & OBERG CJ. 2005. Influence of Calcium, pH, and Moisture on Protein Matrix Structure and Functionality in Direct-Acidified Non-fat Mozzarella Cheese. J Dairy Sci 88(11): 3754-3763.). The elasticity shows if the biochemical reactions taking place inside the cheese are not enough to modify the final structure, giving more or less flexibility. Treated cheeses containing St+Lh were less cohesive at 20 days; that is, they had lower internal bond strength and thus no resistance to structural disintegration.

For the lowest value for the elasticity (Lh + ST on the 20th day of maturation), the moisture showed a decrease of 52.65% at 10 days to 34.61%. Pinho et al. (2004)PINHO O, MENDES E, ALVES MM & FERREIRA I. 2004. Chemical, Physical, and Sensorial Characteristics of “Terrincho” Ewe Cheese: Changes During Ripening and Intravarietal Comparison. J Dairy Sci 87: 249-257. evaluated the texture profile of Terrincho cheese during 60 days of maturation and observed that in the first 20 days of maturation, there was an increase in the gumminess and chewing and a decrease in the adhesiveness, elasticity, cohesiveness of the cheese. According to the authors, the change in texture was attributed to a decrease in pH below 5.5.

The ABTS radical sequestration method measures the antioxidant activity of compounds of hydrophilic and lipophilic nature (Gülçin et al. 2010GÜLÇIN I, BURSAL E, ŞEHITOĞLU MH, BILSEL M & GÖREN AC. 2010. Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. Food Chem Toxicol 48(8-9): 2227-2238., Karadag et al. 2009KARADAG A, OZCELIK B & SANER S. 2009. Review of methods to determine antioxidant capacities. Food Anal Methods 2(1): 41-60.). The annatto seed provided for animals in the diet presents the carotenoid bixin (Bixa orellana L), which has an antioxidant potential (Nozière et al. 2006NOZIÈRE P, GRAULET B, LUCAS A, MARTIN B, GROLIER P & DOREAU M. 2006. Carotenoids for ruminants: From forages to dairy products. Anim Feed Sci Tech 131(3-4): 418-450.), conferring greater reactivity of these molecules to oxidizing agents, thus providing greater stability (Kiokias & Gordon 2013KIOKIAS S & GORDON MH. 2003. Antioxidant properties of annatto carotenoids. Food Chem 83(4): 523-529.).

Carotenoids can be degraded when exposed to light or when subjected to high temperatures during colonial cheese processing, which can influence the antioxidant capacity (Rocha Garcia et al. 2012ROCHA GARCIA CE, BOLOGNESI VJ, GASPARI DIAS JDF, GOMES MIGUEL O & KLOCKER COSTA C. 2012. Carotenoides bixina e norbixina extraídos do urucum (Bixa orellana L.) como antioxidantes em produtos cárneos. Ciênc Rural 42(8).).

During maturation, CLA can be formed from linoleic acid through the action of primary or secondary cultures (Collomb et al. 2003COLLOMB M, MALKE P, SPAHNI N, SIEBER R & BUTIKOFER U. 2003. Dosage des acidesgraslibres dans le fromage par chromatographiegaz-liquide: Précision de la method et differences saisonniéres de la lipolyse dans divers fromages suisses. Travaux de Chemie Alimentary et d’ Hygiéne 94: 212-229.), with a change in lipid profile due to lipolysis reactions. Some bacteria have this characteristic including Butyrivibrio fibrisolvens, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium brevis, Bifidobacterium longum, Propionibacterium acnes, Propionibacterium freudenreichii and Propionibacterium sporogenes (Yang et al. 2017YANG B ET AL. 2017. Bacterial conjugated linoleic acid production and their applications. Prog Lipid Res 68: 26-36.).

A number of CLA-producing species and strains have been reported; however, the mechanisms for CLA bioconversion have not been elucidated for each species, and the individual potential is unknown (Yang et al. 2017YANG B ET AL. 2017. Bacterial conjugated linoleic acid production and their applications. Prog Lipid Res 68: 26-36.).

Lactobacillus reuteri was the first species of lactic acid bacteria related to the high production capacity of CLA (Rosson et al. 1999ROSSON RA, GRUND AD, DENG MD & SANCHEZ-RIERA F. 1999. Linoleate Isomerase. World Patent, WO-99/32604 A1.) besides Lactobacillus plantarum AKU1009 with a conversion rate of up to 85% of linoleic acid (LA) converted to c9, t11-CLA (Kishino et al. 2002KISHINO S, OGAWA J, OMURA Y, MATSUMURA & SHIMIZU S. 2002. Conjugated linoleic acid production from linoleic acid by lactic acid bacteria. J Am Oil Chem Soc 79(2): 159-163.). L. helveticus and S. thermophilus also have the ability to produce CLA according to studies conducted by Pariza & Yang (1999)PARIZA MW & YANG XY. 1999. Method of producing conjugated fatty acid. United States Patent 5856149: 1-12. and Lin et al. (1999)LIN TY, LIN CW & LEE CH. 1999. Conjugated linoleic acid concentration as affected by latic cultures an added linoleic acid. Food Chem 67: 1-5..

According to Ribeiro et al. (2018)RIBEIRO SC, STANTON C, YANG B, ROSS RP & SILVA CC. 2018. Conjugated linoleic acid production and probiotic assessment of Lactobacillus plantarum isolated from Pico cheese. LWT 90: 403-411., CLA values can be related to factors such as temperature, fermentation time, linoleic acid content (LA). Some authors have shown a positive correlation between CLA formation and the ability to tolerate free LA, suggesting that is a detoxification mechanism (Adamczak et al. 2008ADAMCZAK M, BORNSCHEUER UT & BEDNARSKI W. 2008. Properties and biotechnological methods to produce lipids containing conjugated linoleic acid. Eur J Lipid Sci Technol 110(6): 491-504., Wang et al. 2007WANG LM, LV JP, CHU ZQ, CUI YY & REN XH. 2007. Production of conjugated linoleic acid by Propionibacterium freudenreichii. Food Chem 103(2): 313-318.).

Production of CLA in cheeses was also verified by Chamba & Perread (2002)CHAMBA JF & PERREARD E. 2002. Contribution of propionic acid bacteria to lipolysis of Emmental cheese. Lait 82: 33-44., who studied Emmental, and by Woo et al. (1984)WOO AH, KOLLODGE S & LINDAYRC. 1984. Quantification of major free fatty acids in several cheeses varieties. J Dairy Sci 67: 960-968., who studied Roquefort and blue cheeses. Zlatanos et al. (2002)ZLATANOS S, LASKARIDIS K, FEIST C & SAGREDOS A. 2002. CLA content and fatty acid composition of greek Feta and hard cheeses. Food Chem 78: 471-477. reported an increase in CLA with maturation times in hard cheeses when compared to fresh cheeses. Some strains are inhibited by LA, suggesting the need to study specific producer strains, as well as cheese processing conditions and maturation (Sieber et al. 2004SIEBER R, COLLOMB M, AESCHILIMANN A, JELEN P & EYER H. 2004. Impact of microbial cultures on conjugated linoleic acid in dairy products –a review. Int Dairy J, p. 1-15.).

Perotti et al. (2008)PEROTTI MC, BERNAL S, WOLF V & ZALAZAR CA. 2008. Perfil de ácidos grasos libres y características sensoriales de quesos reggianito elaborados con diferentes fermentos. Grasas y aceites 59(2): 152-159. evaluated the free fatty acid profile (C6: 0 to C18:2) at different maturation times with different strains of L. helveticus, which showed no significant differences.

The cheese produced showed high values of saturated fatty acids (SUFA: 63.1508 mg/g fat) similar to those observed by Carafa et al. (2019)CARAFA I, STOCCO G, FRANCESCHI P, SUMMER A, TUOHY KM, BITTANTE G & FRANCIOSI E. 2019. Evaluation of autochthonous lactic acid bacteria as starter and non-starter cultures for the production of Traditional Mountain cheese. Food Res Int 115: 209-218., who evaluated cheeses produced with Lactococcus lactis subsp. lactis, S. thermophilus and Lactobacillus rhamnosus. The higher production of palmitic (14: 0), myristic (16: 0) and stearic (18: 0) fatty acids led to the high values obtained for saturated fatty acids (SUFA) at 7 months of maturation.

The recommended n6:n3 ratio for humans in diets should be 2:1 to 3:1 (Masters 1996MASTERS C. 1996. Omega-3 fatty acids and the peroxisome. Mol Cell Biochem 165(2): 83-93.). Diets based on lower ratios inhibit the transformation of linoleic acid into very long chain polyunsaturated fatty acids (Martin et al. 2006MARTIN CA, ALMEIDA VVD, RUIZ MR, VISENTAINER JEL, MATSHUSHITA M, SOUZA NED & VISENTAINER JV. 2006. Omega-3 and omega-6 polyunsaturated fatty acids: importance and occurrence in foods. Rev Nut 19(6): 761-770.).

In general, cheeses are related to a high concentration of long chain fatty acids; however, it is also known that they contain unsaturated fatty acids, such as oleic acid and CLA, that are important for health. The composition of fatty acids in cheeses varies according to the animal breeds, time of year, animal and species diet and manufacturing processes (González-Martín et al. 2017GONZÁLEZ-MARTÍN MI, PALACIOS VV, REVILLA I, VIVAR-QUINTANA AM & HERNÁNDEZ-HIERRO JM. 2017. Discrimination between cheeses made from cow’s, ewe’s and goat’s milk from unsaturated fatty acids and use of the canonical biplot method. J Food Compos Analysis 56: 34-40.).

CONCLUSIONS

Ripened cheeses produced with L. helveticus and S. thermophilus showed physicochemical characteristics according to the standards for low and medium moisture cheeses. Cheeses containing L. helveticus had lower saturated fatty acid content and higher monounsaturated fatty acid content with a higher PUFA:SUFA ratio.

ACKNOWLEDGMENTS

The present study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), “Fundação Araucaria” – Curitiba, Pr, Instituto Nacional de Ciência e Tecnologia da Cadeia Produtiva do Leite (INCT-Leite UEL).

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Publication Dates

  • Publication in this collection
    30 June 2021
  • Date of issue
    2021

History

  • Received
    13 June 2019
  • Accepted
    25 Oct 2019
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