CHEESE MAKING USING PIG RENNET AND CALF RENNET: MICROORGANISMS AND VOLATILE COMPOUNDS IN FARINDOLA EWE CHEESE

Abstract

Pecorino cheese, a traditional local product of Farindola, is a unique cheese made using pig rennet in Italy. In this study, the evolution of bacterial flora and volatile substances at different ripening times of pig rennet and calf rennet cheese were investigated, taking into consideration the "Production Regulation". The results showed interesting differences between the two types of cheese as a function of volatile substance and microorganism evolution. Gas-Chromatographic analysis showed that, the particular volatile substances profile of cheeses made with pig rennet. Linear Discriminant Analysis was applied, to classify the cheese samples according to different rennets and treatments.

Headnote

ABSTRACT

Pecorino cheese, a traditional local product of Farindola, is a unique cheese made using pig rennet in Italy. In this study the evolution of bacterial flora and volatile substances at different ripening times of pig rennet and calf rennet cheese were investigated, taking into consideration the "Production Regulation". The results showed interesting differences between the two types of cheese as a function of volatile substance and microorganism evolution. Gas-Chromatographic analysis showed the particular volatile substances profile of cheeses made with pig rennet. Linear Discriminant Analysis (LDA) was applied to classify the cheese samples according to different rennets and treatments.

- Keywords: Pig rennet, calf rennet, microorganism ewe cheese, Farindola ewe cheese -

INTRODUCTION

The functionality and the origin of rennet are considered important factors in the making of cheese (BALCONES et al., 1996) and in the production of cheese flavors (URBACH, 1997).

Farindola ewe cheese is a traditional product made using pig rennet and its production takes place in Farindola (Abruzzo) and in some areas of the country in the provinces of Pescara and Teramo. This product comes from an old tradition dating from the Roman period which is called "Vestini cheese". In the scientific literature it seems to be the only known cheese product that is made using pig rennet. The use of pig rennet became obsolete in industrial cheese production because it is not very stable and operates at a pH range that is narrower than that of calf rennet; a standardization to obtain constant enzyme activity in pig rennet is more difficult than in calf rennet.

Farindola ewe cheese is a niche product, the preparation of which is regulated by a "Production regulation", as has been described in previous studies (DI GIACOMO et al, 2009; DI GIACOMO et al, 2014). In these works a comparative study between cheeses made with pig rennet and calf rennet as a function of maturation, amino acids, fatty acids, vitamins, cholesterol evolution and the qualitative determination of some volatile substances was carried out. In addition, a sensory panel of expert tasters showed differences between the two types of cheese: the ewe cheese made with calf rennet is consistently more spicy ("hot" flavor) and more bitter than the ewe cheese made with pig rennet, whereas this latter is always sweeter and never bitter.

One of the most important parameters that effects the judgment of tasters is the flavor of cheese, which depends on the different cheese varieties and on the correct balance and concentration of a wide range of taste and aromatic compounds. Furthermore, the use of raw and unpasteurized milk, increases the flavour notes (GRAPPIN and BEUVIER, 1997) greatly thanks to the different microorganisms naturally present (MARILLEY and CASEY, 2004; GARDE et al., 2002).

This study represents an advanced research of Farindola ewe cheese with the aim of observing the evolution of volatile compounds and the relationships with the microbial flora. In fact, only lower molecular weight compounds contribute significantly to cheese flavor. An important group of low weight molecular compounds are the volatile compounds (SABLE' and COTTENCEAU, 1999). The biochemical pathway for the production of flavour compounds in cheese during their ripening is reviewed by MCSWEENET and SOUSA (2000).

MATERIALS AND METHODS

Cheese making

Fresh ewe milk, coming from ewes that produce less than 1 litre/day in about 100 milking days, was kept cool (10°-12°C). A small quantity of milk was curdled at 31°-33°C with pig rennet and another quantity was processed under the same conditions with calf rennet. The setting time varied from 40 to 60 minutes; after the curdle was broken into granules of 0.5 to 2 cm, it was placed into straw forms to harden; it was then dry salted with coarse salt on both sides - one side of the cheese was salted one day, and the other side the next day. Then the salt was washed off. The period of ripening varied from a minimum of three months to a maximum of nine months and each cheese form weighed between 1 and 2 kg.

Samples and Parameters

1) 2 kg forms were made. They are generally commercialized after 3-6 months, but even up to 1 year; they are ripened at a temperature between 10° and 14°C; 2) Time: the samples were analyzed at 3, 6 and 9 months; 3) the surface of some cheese forms were treated with extra virgin olive oil and vinegar (this treatment is included in the "Production Regulation"), some other were left untreated1.

Microorganism analysis

The growing substrate, the growing temperature and the aerobic/anaerobic conditions are reported in the following scheme.

Volatile compounds analysis

The time evolution of 31 volatile compounds was determined using gas chromatography - mass spectrometry; the extraction methodology of volatile compounds was discussed extensively in an earlier study regarding ewe dairy products (POVOLO et al, 2007). This study can be summarized as follows: a divinylbenzene/ carboxen/polydimethylsiloxane, 50/30 pm, 2-cm-long fiber was used to collect volatile fractions by SPME. 6 g of grated cheese, roughly cut into small pieces shortly before the analysis, was weighed in a crimp-top vial. Cheese samples were allowed to equilibrate to 45°C in a thermostatic bath for 5 min. The extraction of volatile compounds from pasture was performed in duplicate, maintaining the sample at room temperature, and exposing the fiber to the headspace for 15 min. The gas chromatographic analysis of the volatile compounds adsorbed on the SPME fiber was carried out with a CP-WAX 52CB capillary column. A mass spectrometer was used.

Statistical analysis

Linear Discriminant Analysis (LDA) was applied to separate the analyzed cheese samples according to the type of rennet and treatment in order to evaluate the sample differentiation and classification of the data expressed as discriminant scores. All data obtained were analyzed statistically using the multivariate statistical approach through the use of SPSS 8.0 statistical software. This methodology was applied to separate the cheese samples based on the presence of volatile compounds and microorganisms.

LDA has been extensively discussed by several authors (ANDERSON 1984; LEBARTetaZ., 1984; MARDIA et al, 1993).

RESULTS AND DISCUSSION

Microorganism evolution

The microorganism evolution, during the ripening is reported in Table 1. The values are expressed in CFU/g of cheese. Most of the microorganisms show a typical decreasing trend as a function of the ripening. As an example, the trend of lactobacilli, Enterococcus spp., yeasts and moulds is shown in Figs. 1-4.

At starting time, the most important microbiological difference between the cheese made with pig rennet and the cheese made with calf rennet is the content of mesophilic lactobacilli (1.4xl07CFU/gin cheese made using pig rennet compared; 9.6xl06 CFU/g in cheese made using calf rennet), the yeast content (2.7x 105CFU/g in cheese made using pig rennet; 2.9xl06 CFU/g in cheese made using calf rennet), the mould content (2.0x103CFU/g in cheese made using pig rennet; 8.5x104 CFU/g in cheese made using calf rennet) and propionic bacteria content (4.0x102CFU/g in cheese made using pig rennet; 1.6xl03 CFU/g in cheese made using calf rennet).

In the third month of ripening, the cheese made using treated pig rennet has a lactobacilli content of 4.0x107 CFU/g, while the cheese made using untreated pig rennet has a content of 2.7xl07 CFU/g; in the cheeses made using calf rennet the content of lactobacilli is lower: 1.6xl07 CFU/g in treated cheese and 2.0xl07 CFU/g in the untreated cheese respectively. It's interesting to observe that, at this time of ripening, the content of mould in cheese made using untreated pig rennet (3.5xl04 CFU/g) is greater than in other samples (2.5x 102 CFU/g in cheese made using treated pig rennet; 5.Ox lO^FU/g in cheese made using treated calf rennet; l.OxlO3 CFU/g in cheese made using untreated calf rennet).

At six months, the trend of lactobacilli changes completely with regards to the type of rennet. At this time, the lactobacilli content in cheese made with pig rennet decreased: 1.91xl06 CFU/g in the treated cheese and 1.3x 106 CFU/g in the untreated cheese; on the contrary, cheese made using calf rennet at this point in the ripening shows an increase in the lactobacilli content (4.4xl06 CFU/g in the treated cheese; 3.6xl06 CFU/g in the untreated cheese).

At the same time, the yeast content is greater in the cheeses made using pig rennet (1.9xl05 in the treated cheese and 2.3xl05 CFU/g in the untreated cheese) than in cheeses made using calf rennet (2.7xl03 CFU/g in the treated cheese and 5.9xl03 CFU/g of cheese in the untreated cheese).

As regards the evolution of mold, all four types of cheese show the same mold content after six months of ripening.

The citrate fermenting bacteria show the major differences between the samples at six months: a content of 7xl06CFU/g in cheese made using treated pig rennet, 3.4x106CFU/g in cheese made using untreated pig rennet whole the sample made using untreated calf rennet contains only S.ÖxKTCFU/g.

The cheese made using untreated pig rennet has a higher content (2.7xl04 CFU/g) of beta lipolityc bacteria at six months, compared with the other cheese sample (5xl03).

However, these differences observed are not statistically significant at p value less than 0.05.

Quantification and evolution of volatile substances

The volatile compound evolution during the ripening is reported in Table 2. The evolution of volatiles showed a similar trend both in cheeses made with calf rennet and in cheese made with pig rennet. The total volatile compound concentration increases up to six months and then slightly decreases in the next months. The cheeses made with pig rennet are richer in volatile substances, with the exception of some compounds.

At three months of ripening, in the cheese made using treated pig rennet, the volatile substance with the greatest concentration is 2 - butanol (1,463.1 pg/g); it is also present at high levels in cheese made using untreated pig rennet (1,297.4 pg/g) whereas it is present in lower levels in the cheese made using calf rennet (541.2 and 672.1 pg/g for treated and untreated cheese, respectively). The volatile substance, which is most abundant in cheeses, is methyl ethyl ketone (2,636.9 pg/g in untreated pig rennet cheese; 1,618.7 pg/g in treated calf rennet cheese; 1,764.2 pg/g in untreated calf rennet cheese), while the cheese made using treated pig rennet is poorer in this substances (972.9 pg/g of cheese).

The level of acetic acid in cheeses made using pig rennet (575.0 pg/g in treated and 439.1 pg/g in untreated) is higher than cheeses made using calf rennet (300.5 pg/g in treated and 310.6 pg/g in untreated). The content of butyric acid is more than 300 pg/g in all types of cheeses.

After six months of ripening, the volatile compounds profile changes. In the cheese made using pig rennet, butyric acid is the volatile substance present in the highest quantity (3,628.4 pg/g in treated and 2,606.3 pg/g in untreated), a higher level than that found in cheeses made using calf rennet (2,059.4 pg/g in treated and 2,182.2 pg/g in untreated cheese). 2 -heptanone is present at high levels both in cheese made using calf rennet (2,736.9 pg/g in treated and 2.550.6 pg/g in untreated cheese) and in cheese made using pig rennet (2,747.5 pg/g in treated and 2,337.3 pg/g in untreated cheese). Another important substance is hexanoic acid, which is present in higher levels in cheeses made using pig rennet (more than 2,000 pg/g) in comparison with cheeses made using calf rennet. Other volatile substances present in quantities of more than 1,000 pg/g in cheeses made with treated pig rennet are 2 - butanol, methyl ethyl ketone and 2 - nonanone.

The profile of volatile substances differs depending on the type of rennet, time of aging and treatment. The best quality of Farindola Cheese is that made using pig rennet and this is confirmed by the different chemical profiles of volatile substances.

Correlations between volatile compounds and microorganisms

Tables 3 and 4 show the most significant correlations (Pearson) between some volatile substances and microorganisms, at 3 and 6 months of ripening (p < 0.01) for pig rennet and calf rennet cheeses respectively. As can be observed from the correlation (Tables 3 and 4), the evolution of microorganisms and volatile substances is clearly different in the cheeses made with the two types of rennet. In particular, some bacteria are correlated differently with volatile substances as in the following examples that are reported:

a) the mesophilic bacteria are negatively correlated with some substances in cheeses made with calf rennet while they are not correlated in those made with pig rennet; b) Enterococcus spp. are correlated with some substances of cheeses made with pig rennet, while they are not correlated in the cheeses made with calf rennet; c) coccus show a positive correlation with limonene in cheeses made with calf rennet and they show a strong positive correlation with diacetyl in cheeses made with pig rennet; d) the lactobacilli showed a strong positive correlation with diacetyl in cheeses made with pig rennet, while there is a strong negative correlation with ethanol in cheeses made with calf rennet.

Statistical analysis

A significant Wilks Avalué was obtained when the cheese samples were classified as a function of the rennets, using the microorganism evolution data set. In this case, one discriminant function was estimated, since the number of groups in this sample was 2, and 2-1 is the maximum allowable number of eigenvalues for the matrix W 1B. The first discriminant eigenvalue (9.430) had a Wilks A value close to zero (0.096). The distribution of data expressed as discriminant scores along the first eigenvector is presented in Fig. 5. The two sample classes, corresponding to cheese samples with pig rennet and calf rennet, respectively, were clearly distinct. In this case, all cheese samples were correctly assigned to the group they belong to. Furthermore the overall classification success was 100.0%.

According to Wilks A value another distribution was quite significant. In fact, if the data set regarding the volatile substance content is analysed, the results obtained are the following. In this case, one discriminant function was estimated, since the number of groups in this sample was 2, and 2-1 is the maximum allowable number of eigenvalues for the matrix W B. The first discriminant eigenvalue (108.743) had a Wilks A value close to zero (0.009). The distribution of data regarding the volatile substances expressed as discriminant scores along the first eigenvector is presented in Fig. 6. In this representation of all volatile substances, the two sample classes, corresponding to cheese samples as function of the two rennets, respectively, were distinct and the overall classification success was 100.0%.

A significant Wilks Avalué was obtained when the data set regarding the volatile substances was classified as a function of treatments. In this case, one discriminant function was estimated; the first discriminant eigenvalue (20.227) had a Wilks A value close to zero (0.047). The distribution of data regarding the volatile substances as a function of treatment expressed as discriminant scores along the first eigenvector is presented in Fig. 7. In this representation of data regarding the volatile compounds, the two sample classes, corresponding to cheese samples as function of the treatments, respectively, were clearly distinct. In fact the overall classification success was 100.0%.

A significant Wilks Avalué was obtained when the cheese samples were classified as a function of the rennets and of the treatment, using a volatile substance data set. In this case, 3 discriminant functions were estimated, since the number of groups in this sample was 4, and 4-1 is the maximum allowable number of eigenvalues for the matrix W 1B. The first discriminant eigenvalue (27.979) had a Wilks A value close to zero (0.005). The distribution of data expressed as discriminant scores along the first two eigenvectors is presented in Fig. 8. In this representation of all data, the four sample classes, corresponding to cheese samples with treated pig rennet (1), untreated pig rennet (2), treated calf rennet (3) and untreated calf rennet (4), respec- tively, were clearly distinct. It may also be noted that the centroids of each group fall into different quadrants.

In this case, all cheese samples were correctly assigned to the group they belong to. Furthermore the overall classification success was 100.0%.

Panel test

Fig. 9 shows sensorial differences analyzed by expert tasters between cheese made with calf rennet and pig rennet (sweeter, less bitter and less spicy) in the same production condition (DI GIACOMO et al, 2009, DI GIACOMO et al, 2013).

CONCLUSIONS

It can be observed that there is a different microorganism evolution depending on the type of rennet, especially as regards lactobacilli, Enterococcus spp. and yeast. It follows that the volatile compounds profile is different between the cheese made with calf rennet and the cheese made with pig rennet. Pearson correlations between micro - organisms and volatile compounds show differences as a function of type of rennet.

These differences influence the aromatic profile, as confirmed in a study carried out by a panel of experts who judged the Farindola ewe cheese sweeter, less bitter and less spicy than ewe cheese made with calf rennet in the same production conditions. Probably consumers prefer Farindola ewe cheese for these reasons.

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Paper received July 9, 2013 Accepted October 25,2013