Amino acid profile and sensory characteristics of dry fermented pork ...

Research Article Received: 4 January 2016

Revised: 12 October 2016

Accepted article published: 9 November 2016

Published online in Wiley Online Library: 7 December 2016

(wileyonlinelibrary.com) DOI 10.1002/jsfa.8133

Amino acid profile and sensory characteristics of dry fermented pork loins produced with a mixture of probiotic starter cultures a* Anna Okon, a and ̇ ´ ´ b Danuta Kołozyn-Krajewska Katarzyna Neffe-Skocinska, Zbigniew Dolatowskib

Abstract BACKGROUND: Proteolysis is a biochemical process in dry-aged meat products where proteins are metabolized and broken down to polypeptides, peptides, and free amino acids. In the literature it is reported that an appropriate choice of probiotic starter culture limits proteolytic changes in dry-fermented meat products. In this study the combined effect of a mixture of probiotic starter cultures on the free amino acid profile, total count of lactic acid bacteria, and the sensory quality of dry-aged pork loins after fermentation and after storing the vacuum-packed samples was evaluated. RESULTS: LOCK900 and BB12 probiotic strains were the technologically best two-species mixture of starter cultures for the production of probiotic dry-aged pork loins. They allowed us to obtain products with high and stable bacterial count and acceptable sensory quality, both after 21 days of fermentation and after 2 months of cold storage. Changes in the free amino acid profile and increased intensity of the selected sensory attributes result from a significant share of probiotics in meat proteolysis occurring during fermentation and storage. CONCLUSION: The results suggest the relevance of using probiotic bacteria as a two-species starter culture for the production of dry-aged products. © 2016 Society of Chemical Industry Keywords: probiotic starter cultures; lactic acid bacteria; free amino acids; proteolysis; sensory quality; dry-aged loins

INTRODUCTION

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growth of pathogenic microflora and adverse physical and chemical changes of lipids and proteins. In addition to their ability to limit the metabolic rate of lipid oxidation, probiotics have the ability to inhibit proteolytic reactions that occur in the product during storage.5 Proteolysis is one of the most important biochemical processes in dry-aged meat products, whereby proteins are metabolized and broken down into polypeptides, peptides, and free amino acids.6,7 Protein breakdown takes place with the participation of endogenic and microbiological enzymes, which in turn often leads to deamination and decarboxylation. The basic products of protein decarboxylation in meat are biogenic amines: cadaverine, putrescine, tyramine, and histamine. Previous studies have shown that the appropriate choice of probiotic strains limits proteolytic changes in dry-fermented meat products.8,9

∗

Correspondence to: K Neffe-Skocińska, Department of Food Gastronomy and Food Hygiene, Warsaw University of Life Sciences (WULS), Nowoursynowska St 159C, 02–776 Warsaw, Poland. E-mail: katarzyna_neff[email protected]

a Department of Food Gastronomy and Food Hygiene, Warsaw University of Life Sciences (WULS), 02-776 Warsaw, Poland b Department of Meat Technology and Food Quality, University of Life Sciences (LULS), 20-704 Lublin, Poland

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© 2016 Society of Chemical Industry

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FAO/WHO defines probiotics as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.1 Numerous studies of the gut microflora suggest that probiotic lactic acid bacteria (LAB) from food are able to colonize the intestine and thus play an important role in its proper functioning. Probiotic products should contain not less than 6.0 log CFU g− 1 (CFU mL−1 ) of these microorganisms until the end of its shelf life. This level is called the ‘minimum therapeutic’ concentration. Probiotics play an important role in the protection of the host against harmful microorganisms and also strengthen the immune system. They must be safe, acid and bile tolerant, and able to adhere to and colonize the intestinal tract.2 Starter cultures are live, defined, and specially selected microorganisms with GRAS (generally recognized as safe) safety status, responsible for the desired course of meat fermentation and aging. Their use in the production of dry-fermented meats is always intentional and aims at obtaining the specified sensory and microbiological characteristics in the end product. Currently, the production of commercial starter preparations using primarily LAB shows a favorable technological effect.3,4 The important aspect of using a probiotic starter culture in the production of dry-fermented meats, in addition to the possibilities of growth and survival in the meat environment and exercising a favorable effect on human body, is the ability to inhibit the

´ K Neffe-Skocinska et al.

www.soci.org Thus, based on our previous studies and literature data, it is possible to hypothesize that there is the possibility of using selected strains of probiotic bacteria as a starter culture in the manufacture of dry-aged pork loins. In addition to further developing this hypothesis, the combined effect of a mixture of probiotic starter cultures on the free amino acid profile, total count of LAB, and the sensory quality of dry-aged pork loins after fermentation and after storage of vacuum-packed samples was evaluated.

MATERIALS AND METHODS Starter culture preparation Two known probiotic strains that fulfilled the required criteria, and one potential probiotic strain, were used in this study: • The probiotic strain Lactobacillis rhamnosus LOCK90010 (Patent No. P-382760) was from the collection of the Technical University of Łód´z, Poland. In the present study, specific attention was paid to LOCK900, which demonstrated the best technological characteristics in our previous microbiological, physicochemical and sensory tests appropriate for the production of dry-fermented meats.6,16,17 The probiotic strain Bifidobacterium animalis subsp. lactis BB12 was from the Chr. Hansen collection. • The potential probiotic strain Lactobacillus acidophilus was from the collection of the Technical University of Łód´z, Poland. Each of the starter cultures was prepared separately. The process of preparing the probiotic starters involved two stages: • Boost process: 5 mL MRS broth was inoculated with probiotic strain and incubated for 24 h at 37 ∘ C. • Preparation process of probiotic starter: after 24 h the tubes were centrifuged for 5 min at 10 000 × g (laboratory centrifuge MPW-251; MPW MED. Instruments, Warsaw, Poland) to separate the cells of the probiotic strain from the growth medium. MRS broth was replaced with a special food nutrient medium to allow further growth of bacteria for 24 h at 37 ∘ C (subject of patent). In the prepared starter culture the number of probiotic bacteria was determined using a Tempo System (bioMérieux, France). The count of probiotic bacteria was 9.0 log CFU mL−1 . Starter cultures were combined in a sterile beaker immediately prior to addition to the raw meat.

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Loin production and sampling procedures This study was carried out in strict accordance with the Polish legal guidelines for the protection of animals for slaughter and use, edited by the Polish Ministries of Agriculture and Rural Development. The pork loin was chilled to 7 ∘ C and divided into parts 48 h post mortem. Loin samples were cured using the ‘dry’ method with the curing mixture (sea salt 20 g kg−1 loin, curing salt 9.7 g kg−1 loin, and NaNO3 0.3 g kg−1 loin) in a quantity of 28 g kg−1 . The curing process was conducted for 48 h at 0 ∘ C. The three different mixtures of starter cultures (2 mL kg−1 meat, containing 6.0 log CFU mL−1 ) and glucose (5 g kg−1 meat) were then added to each loin sample. Subsequently, loins were fermented at 16 ∘ C for 21 days in a fermentation chamber with a relative humidity of between 70% and 75%. After 10 days the loins were cold smoked (30 min, 30 ∘ C). After 21 days of fermentation the samples were vacuum packed and stored at 4 ∘ C for 60 days. Microbiological, chemical, and sensory analyses were carried out after every 30 days of storage time. In this study two research

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moments for changes in free amino acids profile were selected and presented: after 21 days of fermentation and 60 days of storage. Two series of experiments were performed. Five kinds of sample were produced: • C: control loin without probiotic starter cultures; • C-LOCK900: control loin with one probiotic strain – LOCK900; • LOCK900 + Bauer: mixture of two probiotic strains – LOCK900 and Bauer; • LOCK900 + BB12: mixture of two probiotic strains – LOCK900 and BB12; • Bauer + BB12: mixture of two probiotic strains – Bauer and BB12. Microbiological analysis Microbiological analyses were carried out using the Tempo System (automated quality indicator solution; bioMérieux, France) and Tempo LAB tests (automated test for the enumeration of LAB microorganisms; bioMérieux, France). Calculation of bacteria number in the study samples (log CFU g−1 ), according to the Tempo System, is based on the most probable number (MPN) method. The Tempo LAB test was able to obtain performance levels similar to the traditional plate method and to the standard ISO 15214.11 Samples for microbiological analysis were prepared in accordance with Jaworska et al.12 Incubation parameters were 48 h and 37 ∘ C. Amino acid analysis A 15 g sample was mixed with 3% salicylic acid to 200 mL. The mixture was homogenized and centrifuged for 15 min at 3000 × g. The prepared sample was dispensed onto the column for amino acid analysis, which was followed by separation. The individual analytes were changed at derivatives thereof in a reactor using colored complex amino-ninhydrin. Identification was carried out in a photometric detector. The values were expressed in milligrams per gram of product. The amino acid composition was determined by ion-exchange chromatography (amino acid analyzer AAA 400, INGLOS Co.). The test was performed at the Central Laboratory Agro-Ecological University of Life Sciences in Lublin (Poland). Sensory analysis The sensory QDA method was used.13 Descriptors were chosen and defined during a panel discussion and then verified in a preliminary session. Sixteen sensory attributes were measured to quantify the quality of the tested products (Table 1). An unstructured, linear graphical scale of 100 mm was afterwards converted to numerical values (0–10 conventional units, c.u.). The marks of anchors of the tested attributes were for most of them as follows: none–very strong, also for meat colour (light–dark), homogeneity of colour (none–very high) and for juiciness (dry–juicy). On the basis of the above-mentioned quality characteristics, the assessing sensory panel indicated an overall sensory quality (low–very high) for each sample on a separate scale. The preparation of samples for QDA analyses were made in accordance with Jaworska et al.12 Statistical analyses All statistical analyses were performed using Statistica software (version 8.0, StatSoft, Inc.). Analysis of variance (ANOVA) was employed to determine any significant difference among samples. Values were considered significantly different when P < 0.05.


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Effect of probiotic strains on free amino acid profile in dry-aged loin

Table 1. Definition of the sensory attributes Attribute Odor 1 Smoked meat 2 Dried meat 3 Sharp 4 Stored, rancid 5 Other Appearance and texture 6 Meat color 7 Homogeneity of color 8 Juiciness

Flavor 9 Smoked meat 10 Dried meat 11 Salty 12 Bitter 13 Stored, rancid 14 Stinging 15 Sour 16 Other Overall quality

Definition

Typical of dry-fermented meat Typical of dry-fermented meat Irritating impression when smelling Off-flavor associated with changes in fat oxidation; lack of freshness Other sensation, not on list Intensity of red color associated with the meat Uniform distribution of red color typical of dry-fermented meat Perception of the amount of water released by the product during mastication Typical of dry-fermented meat Typical of dry-fermented meat Basic quality of taste Basic quality of taste Lack of freshness Basic quality of taste Basic quality of taste Other sensation, not on list Attribute of total quality of dry-fermented pork loin

RESULTS AND DISCUSSION

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previous scientific reports.8,12,16,17 The mixture of two-species probiotic starter cultures using LOCK900, BB12, and Bauer in the production of dry-aged meat products was not examined. Analysis of the results of microbiological examination of pork loins after 21 days of fermentation show clearly that the addition of probiotic strains had a significant effect on the overall LAB count in the product (Fig. 1). After fermentation, the highest LAB count (7.66 log CFU g−1 ) was found in the sample of pork loin with added mixture of LOCK900 and BB12 strains. After the period of cold storage under anaerobic conditions, lasting for 60 days, a statistically significant decrease in this count was observed, on average by one logarithmic unit, but it still remained high (6.92 log CFU g−1 ). The mixture of LOCK900 and BB12 strains proved the best variant of the two-species probiotic starter culture in the production of dry-aged pork loins, with regard to the bacterial count. In samples with added mixture of LOCK900 and Bauer strains, the average LAB count observed after the fermentation period was 5.0 log CFU g−1 . After the storage period, a statistically significant – with regard to time – increase in the LAB count by two logarithmic units (7.75 log CFU g−1 ) was observed. According to Ruiz-Moyano et al.,14 probiotic starter cultures have to adapt well to the conditions in dry-aged meats and to become their dominant microflora. The results of the microbiological analysis of the sample containing LOCK900 + Bauer strains show clearly that L. acidophilus Bauer used in combination with another starter culture does not show good technological characteristics. It was observed that when mixed with L. rhamnosus LOCK900 bacteria, it disturbed the adaptation of LOCK900 strain to the conditions prevailing in raw meat material. This resulted in a low LAB count at baseline in this sample at the end of the fermentation process and a statistically significant increase in this count during storage (Fig. 1). The worst variant of the two-species probiotic starter culture was the mixture of Bauer and BB12 bacterial strains. After both the fermentation and storage period the LAB count, including probiotic strains, did not change significantly and was on average 5.0 log CFU g−1 . Low LAB count, including probiotic strains, indicates the absence of characteristics required for the colonization of raw meat material rich in environmental microflora. This shows that Bifidobacterium sp. bacteria are destroyed during the production and storage of fermented meat products as they are not adapted to the specific conditions of the medium, which is raw meat material. On the other hand, the study conducted by Sionek et al.,16 where single-species probiotic starter cultures (not mixed with each other strains of BB12 and Bauer) were used in the production of dry-fermented sausages, demonstrated very good growth and survival of B. animalis subsp. lactis BB12 bacteria (8.0–9.0 log CFU g−1 ), and poor activity of the potentially probiotic L. acidophilus Bauer. In turn, in the study conducted by Jaworska et al.,12 bacterial strain Bauer was also added as a single-species culture, but to dry-fermented pork loins; it showed good growth, at an average level of 7.00 log CFU g−1 , both after fermentation and 6 months storage. Our studies have demonstrated that the Bauer and BB12 strains used as two-species starter culture (a mixture of them) were not able to present sufficient competition for the microflora naturally living in raw meat material, or competed with each other, which could have prevented their growth in the examined product. On the other hand, this study confirms the importance of the appropriate selection of starter cultures and their technological properties to the raw material or final product. Not without significance is the consistency of the product, such as minced meat in sausages or whole muscle as loin or ham. The varying consistency of individual muscles can create technological problems.18



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Raw meat material is characterized by environmental natural bacterial microflora which are impossible to eliminate prior to commencement of the production process.14 The composition of this microflora in large part contains LAB, as meat is their natural environment and ensures optimal conditions for their growth and development.15 The results of the conducted microbiological tests allowed determination of the overall count of LAB naturally occurring in the examined loins. This count in the control sample (C) remained at a stable level of 4.0 log CFU g−1 both after the completion of the fermentation process and after 60 days of storage in a cold room under anaerobic conditions (Fig. 1). The tests took into account also the comparison of the control sample with added single-species starter culture based on L. rhamnosus LOCK900 (C-LOCK900) (Fig. 1). The mean LAB count immediately after fermentation was 7.0 log CFU g−1 . After the period of cold room storage this count decreased significantly by two logarithmic units, to 5.0 log CFU g−1 . In the study conducted earlier, dry-aged meat with LOCK900 addition demonstrated good microbiological, physicochemical and sensory properties.8,16 Additionally, the LOCK900 probiotic strain in dry-aged pork loins during fermentation and storage was identified using a genetic approach ´ such as the 16S rRNA gene sequencing method (Neffe-Skocinska K, unpublished). The target microbiological analyses were aimed at the assessment of the growth and survival of two-species probiotic starter cultures in pork loins. Each culture contained two different strains. Three variants of starter cultures added to raw meat material were created. The technological characteristics (good microbiological, physicochemical and sensory properties) of the selected probiotic strains used as single-species starter cultures were confirmed in

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Count of LAB [log cfu g-1]

after ripening 9 8 7 6 5 4 3 2 1 0

after storage 7.752C

7.041b

4.221a

7.661b

6.922D 5.102B

5.131c

5.251c

5.071B

4.331A

C

C-LOCK900

LOCK900+Bauer LOCK900+BB12

Bauer+BB12

Pork loins with mixture of probiotic starter cultures added Figure 1. The number of LAB in samples of dry-aged pork loins with or without a mixture of probiotic starter cultures added after 21 days of ripening and after 60 days of storage. Values for the same sample not followed by a common number are significantly different (P < 0.05), for differences for the same sample between ripening, storage, and number of bacteria. Values for samples after ripening not followed by a common lower-case letter are significantly different (P < 0.05), for differences between samples after the ripening process and number of bacteria. Values for samples after storage not followed by a common upper-case letter are significantly different (P < 0.05), for differences between samples after storage and number of bacteria.

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Table 2 reports the content of free amino acids after 21 days of fermentation. Analysis of the free amino acid profile in loins immediately after the fermentation process indicates that the largest number of products of protein changes is characteristic for the control sample, which contains only the natural environmental microflora, and the sample with the two-species starter culture LOCK900 + BB12. Analysis of the free amino acid profile carried out after fermentation demonstrated the highest content of alanine, citrulline, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, tyrosine, and aspartic acid in the sample vaccinated with the two-species starter culture LOCK900 + BB12. Also, after fermentation this investigated variant demonstrated the highest LAB count (Fig. 1) and the highest sensory quality (Fig. 2). Table 3 reports the content of free amino acids after 60 days of storage. After this time an increase in the free amino acid content in all samples was noted, with the highest values observed in the case of the control sample and the sample vaccinated with LOCK900 and Bauer cultures. The highest values of alanine, 𝛽-alanine, citrulline, glycine, histidine, isoleucine, leucine, serine, tyrosine, and valine were noted in the sample containing the mixture of LOCK900 and Bauer strains. At the same time, after the storage period, the highest LAB count was observed in this sample for all examined loin variants (Fig. 1). On the other hand, the sample with the mixture of Bauer and BB12 strains contained seven free amino acids (alanine, arginine, isoleucine, lysine, methionine, threonine, tyrosine) in the lowest concentration after 60 days of storage. Analysis of all obtained results indicated that the highest increase in the levels of all free amino acids during the 2-month storage period was observed in the LOCK900 + Bauer sample (9.4 mg g−1 ), while the lowest increase (0.18 mg g−1 ) was observed in the LOCK900 + BB12 sample. Moreover, analysis of the free amino acid profile immediately after the fermentation of loins in the control group and with the addition of LOCK900 and BB12 strains demonstrated the highest content of glutamic acid (C = 1.09 mg g−1 and LOCK900 + BB12 = 1.05 mg g−1 ) and, in the case of the LOCK900 + BB12 sample, high alanine and tyrosine content (1.15 and 0.51 mg g−1 ). High alanine and tyrosine content (1.24 and 0.37 mg g−1 ) as compared with the control sample was also found in loins vaccinated with the mixture of LOCK900 and Bauer


cultures (1.45 and 0.67 mg g−1 ). After 60 days of cold storage under anaerobic conditions, a much higher level of glutamic acid in the obtained free amino acid content was observed only in the samples vaccinated with probiotic bacteria, as compared with the control sample without the addition of starter cultures. The highest arginine content in the comparative sample with addition of the single-species LOCK900 starter culture (0.43 mg g−1 ) immediately after the fermentation process is also noticeable. This value was nearly twofold higher than in the control sample (0.23 mg g−1 ). After 60 days of storage, the arginine content was also highest in the sample containing LOCK900 strain (0.41 mg g−1 ) and nearly fourfold higher than in the control sample (0.15 mg g−1 ). On the other hand, the highest arginine content was observed in the sample containing the mixture of Bauer and BB12 strains (0.09 mg g−1 ). The probiotic strain used in this study as single or two species changed the qualitative and quantitative levels of amino acids. The important factor in these changes may be the inhibition of the activity of natural environmental microflora present in the raw meat material.19 Bifidobacterium and Lactobacillus types used in the study conditions of the technological process require for their growth the development of certain amino acids, i.e., arginine, isoleucine, leucine, tyrosine, cysteine, and valine.20 In the obtained study results these amino acids are present as free amino acids or in low-molecular-weight peptides. Belkaaloul et al.21 suggested that L. acidophilus Ki bacteria hydrolyze milk proteins to low-molecular-weight peptides that can be used to increase bacterial growth and acidify the environment, which in turn results in the increased count of other bacterial species or strains, i.e., Bifidobacterium animalis ssp. lactis Bo. Similar interactions were observed among other Bifidobacterium strains and the proteolytic Lactobacillus.22 These findings have been confirmed by Aro Aro et al.,23 who did not observe any significant differences in product acidity in a sample comparison after 21 days of fermentation of sausages with various bacterial species (Lactobacillus sakei D-1001, Staphylococcus carnosus SB-61, Staphylococcus xylosus, and Pediococcus pentosaceus), whereas they observed a significant increase in the content of free amino acids in these samples. The content of free amino acids in the products is affected to a large extent by the speed of their degradation to biogenic amines by appropriate enzymes; in turn, the activity of these enzymes is limited by the


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Effect of probiotic strains on free amino acid profile in dry-aged loin

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Table 2. Content of free amino acids after 21 days of fermentation (mg g−1 ) After fermentation Amino acid Ala Arg 𝛽-ala Citr Gama Gly His Ile Glu Leu Lys Met Orn Phe PheSer Pro Ser Tau Thr Tyr Val 1mHis Cys Asp Urea* Total

C

C-LOCK900

0.94 0.23 0.00 0.18 0.17 0.64 0.37 0.47 1.09 0.83 1.45 0.30 0.44 0.44 0.00 0.53 0.48 0.36 0.49 0.32 0.69 1.11 0.00 0.00 3.59 15.12

LOCK900 + Bauer

0.98 0.43a 0.00 0.14a 0.06a 0.61 0.35a 0.45 0.60 0.77 1.41 0.30 0.34 0.41 0.02 0.51a 0.36a 0.39 0.42 0.37 0.62 1.27 0.00 0.00 3.06 13.87

LOCK900 + BB12

0.85 0.18bc 0.50a 0.32b 0.02b 0.53 1.06b 0.54 0.96 0.90 1.61 0.34 0.24 0.50 0.00 0.20b 0.77b 0.32 0.30 0.41 0.66 1.16 0.00 0.00 0.00 12.37

1.15 0.15b 0.38 0.39b 0.04 0.70 0.92b 0.75 1.05 1.26 2.00 0.47 0.42a 0.69 0.00 0.41 1.07b 0.37 0.42 0.51 0.96 1.33 0.00 0.12a 0.00 15.56

Bauer + BB12 0.90 0.30 ac 0.22b 0.15a 0.00 0.47 0.58 0.50 0.76 0.80 1.35 0.29 0.19b 0.45 0.00 0.52a 0.76b 0.30 0.30 0.39 0.63 1.43 0.00 0.04b 0.00 11.33

* Amino acid degradation product. Values for samples after ripening not followed by a common letter are significantly different (P < 0.05).

overall quality

smoked o. 10

dried meat o.

8

other f.

acid o. C

6 sour f.

off-flavor o.

4

C-LOCK900 2 stinging f.

other o. 0

LOCK900+Bauer

storage, rancid f.

color int. LOCK900+BB12 color homog.

bitter f. salty f. dried meat f.

Bauer+BB12

juiciness smoked f.

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Figure 2. Sensory profiles of the dry-aged pork loins after 21 days of fermentation. o, odor; int., intensity; homog., homogeneity; f., flavor. Sensory attributes that significantly impact on the overall quality of dry-aged pork loins are: sample C – smoked o., dried meat o., juiciness, smoked f., dried meat f., salty f.; sample C-LOCK900 – smoked o., dried meat o., color homog., dried meat f.; sample LOCK900 + Bauer – dried meat o., acid o., off-flavor o., color int., dried f., sour f.; sample LOCK900 + BB12 – dried meat o., color homog., dried meat f., salty f., sour f.; sample Bauer + BB12 – dried meat o., juiciness, dried meat f., sour f.


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Table 3. Content of free amino acids after 60 days of storage (mg g−1 ) After storage Amino acid Ala Arg 𝛽-ala Citr Gama Gly His Ile Glu Leu Lys Met Orn Phe PheSer Pro Ser Tau Thr Tyr Val 1mHis Cys Asp Urea* Total

C 1.24 0.15BC 0.00 0.12A 0.67A 0.78 0.44A 0.64 0.48A 1.10 1.87 0.39 0.68 0.58 0.00 0.61B 0.57B 0.50B 0.60B 0.37 0.86 1.25 0.00 0.00 5.15B 19.05

C-LOCK900 1.17 0.41 AC 0.00 0.10A 0.32B 0.72 0.39A 0.55 1.08B 0.92 1.69 0.34 0.43B 0.48 0.00 0.53 0.60B 0.56B 0.57B 0.39 0.73 1.29 0.00 0.00 4.45B 17.72

LOCK900 + Bauer 1.45 0.28ABC 0.80A 0.47B 0.07 1.13 1.46B 0.94 1.36B 1.90 2.51 0.60A 0.44B 0.86 0.00 0.29A 1.57A 0.42B 1.40A 0.67A 1.21 1.31 0.00 0.00 0.60A 21.77

LOCK900 + BB12 1.09 0.14BC 0.39B 0.33B 0.05 0.77 1.11B 0.74 1.38B 1.22 1.78 0.44 0.59 0.78 0.00 0.48 1.11 0.15A 0.53B 0.47 1.10 0.98 0.00 0.11 0.00 15.74

Bauer + BB12 0.99 0.09B 0.51B 0.29B 0.06 0.63 1.00B 0.53 1.56B 1.12 1.67 0.27B 0.99A 0.54 0.00 0.61B 0.93 0.26 0.37B 0.30B 0.89 1.14 0.00 0.12 0.00 14.96

* Amino acid degradation product. Values for samples after storage not followed by a common letter are significantly different (P < 0.05).

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content of table salt and low water activity.24 In the studies conducted by Pomponio et al.,25 a similar water loss was observed in all samples. This results in an increased share of table salt, which inhibits the activity of aminopeptidases; as a result, the quantity of peptides decreases. Immediately after fermentation the content of urea, as the product of amino acid degradation, was highest in the control sample (3.59 mg g−1 ) and the sample containing one strain, LOCK900 (3.06 mg g−1 ). On the other hand, urea was not found in the other variants of samples with the mixture of probiotic bacterial strains. After 2 months of storage the content of urea was highest in the control sample (5.15 mg g−1 ) and again in the sample containing LOCK900 strain (4.45 mg g−1 ), whereas no urea was found in LOCK900 + BB12 and Bauer + BB12 samples. In the present and previous studies an excessive breakdown of amino acids took place in the control sample, which is evidenced also by poorer sensory attributes and poorer sensory quality of the product.6 This shows that there is a dependence between the bitter flavor of dry-fermented meat products and the high activity of cathepsin B at the time of proteolytic changes. The results indicated also that some amino acids, i.e., leucine, isoleucine, phenylalanine and Gly-Phe, Gly-Leu, Gly-Ile, and Ile-Leu dipeptides can be used as markers of an undesired bitter flavor. On the other hand, the addition of glucose applied in the technology is of high significance, not only from the perspective of microbiology and achieving a high LAB count, but also in the context of sensory quality and proteolytic changes occurring in the product. In the presented


studies, the authors noted very low intensity (below 1 c.u.) of sensing bitter flavor in all examined pork loin variants, with the exception of LOCK900 + Bauer sample, where after 60 days of storage a bitter flavor was sensed with twice as high intensity, which could also be connected with a significant increase in the LAB count. High levels of glutamic acid and alanine in probiotic samples observed in the study had a positive impact on the sensory perception of the evaluators. Similar dependences were observed by Casaburi et al.4 in sausages containing Staphylococcus xylosus and Lactobacillus curvatus strains, They demonstrated that L. curvatus positively affects the development of large quantities of glutamic acid, alanine, histidine, arginine, and lysine, while L. sakei affects the levels of glutamic acid, alanine, 𝛾-aminobutyric acid, threonine, leucine, phenylalanine, and ornitine. Other studies confirmed similar changes in the levels of free amino acids in the compositions of traditional fermented sausages, caused by starter cultures with a defined bacterial composition.4,9,19 On the other hand, the share of the applied starter cultures from LAB species, and in particular probiotic species, in the proteolysis of unground meat products has not been fully examined and is in the investigation phase. Figure 2 shows the sensory profiles of the dry fermented pork loins after 21 days of fermentation. Immediately after this time the overall quality of all dry fermented pork loins with the addition of probiotic starter cultures was high. Pork loins with the starter culture composed of L. rhamnosus LOCK900 and B. animalis subsp. lactis BB12 were given the highest score (approximately 7


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Effect of probiotic strains on free amino acid profile in dry-aged loin smoked o. 8

overall quality other f.

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dried meat o. acid o.

6

C 4

sour f.

off-flavor o. C-LOCK900

2 stinging f.

other o. 0

LOCK+Bauer

storage, rancid f.

color int. LOCK+BB12 color homog.

bitter f. salty f.

Bauer+BB12

juiciness

dried meat f.

smoked f.

Figure 3. Sensory profiles of the dry-fermented pork loins after 60 days of storage. o, odor; int., intensity; homog., homogeneity; f., flavor. Sensory attributes that significantly impact on the overall quality of dry-aged pork loins are: sample C – acid o., off-flavor o., juiciness, smoked f., salty f.; sample C-LOCK900 – dried meat o., off-flavor o., color int., color homog., dried meat f., sour f.; sample LOCK900 + Bauer – dried meat o., acid o., off-flavor o., color int., color homog., smoked f., salty f., bitter f., storage f., sour f.; sample LOCK900 + BB12 – color int., juiciness, smoked f., sour f.; sample Bauer + BB12 – dried meat o., off-flavor o., color homog., smoked f., sour f.

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culture selection. This process is the result of balancing the quantities of volatile compounds (alcohol, ketones, aldehydes, furans) and non-volatile compounds (amino acids, peptides, nucleotides, lactate, acetate, butyric acid), produced as a result of saccharide fermentation processes and other metabolic changes in microorganisms added to the product in the form of selected starter cultures.15,20 Non-volatile compounds lend to sausages a characteristic, complex flavor, described in sensory studies as the combination of sour, salty, bitter, and sweet aftertastes.15 While choosing probiotic strains for use in the production of meat products, not only their technological characteristics but also their ability to create the desired flavor and odor should be taken into account.26

CONCLUSIONS It has been proved that Lactobacillus rhamnosus LOCK900 and Bifidobacterium animalis subsp. lactis BB12 probiotic strains were the technologically best two-species mixture of starter cultures for the production of probiotic dry-fermented pork loins. They allowed us to obtain products with high and stable LAB count and acceptable sensory quality, both after 21 days of fermentation and after 2 months of cold storage. Sensory analysis demonstrated that loins with added probiotic strains are acceptable in sensory terms, both after the process of fermentation and after storage. The addition of strains with probiotic properties has a favorable effect on sensory changes occurring in fermented meat products. In addition, changes in free amino acid profile and increased intensity of selected sensory attributes indicate the significant share of probiotics in meat proteolysis during fermentation and storage. The results suggest the relevance of using probiotic bacteria as two-species starter cultures for the production of dry-fermented products.

ACKNOWLEDGEMENTS The strains L. rhamnosus LOCK900 and L. acidophilus Bauer were kindly provided by Professor Zdzisława Libudzisz and Dr Ilona Motyl (Technical University of Łód´z, Poland).



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c.u.). The same sample after 21 days of fermentation contained the highest LAB count, including probiotic bacteria (7.66 log CFU g−1 ). Loins vaccinated with single-species starter culture, i.e., LOCK900, were assessed similarly. After 60 days of cold storage of all vacuum-packed loin samples, the overall quality statistically significantly decreased as compared with scores given immediately after the fermentation process (Fig. 3). In the control sample without added probiotic starter cultures, the overall quality was given the poorest score both after fermentation and after cold storage under anaerobic conditions. After 21 days of fermentation the average overall quality was 6.9 c.u., while after 60 days of storage it was 6.3 c.u. This change was not statistically significant. Statistically significant differences were observed only with regard to the sensory attribute of juiciness (Fig. 3). During all study periods, the control sample was the driest and hardest while chewing, compared to all other variants of loins with probiotic addition. The evaluators on the basis of these sensory feelings can decided with the lowest overall quality of control sample. Changes of juiciness in dry-aged meat products are associated with the loss of water during storage. In the samples of dry-aged loins with the addition of probiotic starter cultures, changes were not limited to the level of juiciness. The changes also included sensory attributors of odor, flavor, and color. Significant changes that occurred during storage in the control sample without the addition of probiotics concerned only juiciness. Therefore, the addition of LAB with probiotic properties for pork loins could be affected by changes in the sensory attributors of odor, flavor, and color during storage. Summing up the results of sensory analysis it can be concluded that pork loins with the addition of various probiotic bacteria were assessed more highly by the expert panel as compared with the control sample (without added starter cultures). Fadda et al.20 and Leroy et al.3 also observed that LAB had a favorable effect on the sensory attributes of dry-fermented meat products. The choice of appropriate LAB strains impacts the formation of the characteristic flavor and odor of fermented meat products.15 The characteristic flavor and smell of dry-aged products is shaped during the properly conducted process of fermentation, aging, and starter


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J Sci Food Agric 2017; 97: 2953–2960