FORMING THE TENDERNESS OF BEEF USING OF SELECTED ENZYMATIC PREPARATIONS MICROBIOLOGICAL ORIGIN

Grażyna Krasnowska, Ewa Rudownik, Andrzej Hyliński, Anna Nowak
Department of Animal Products Technology and Quality Management, Agricultural University of Wrocław, Poland

ABSTRACT

Tenderness of meat qualifies the cooking quality of meat. This parameter results from the structure of meat that depends of many pre-slaughter elements (genotype, age, gender) Functional properties and increasing of water-absorbing creates as results of post mortem transformations of meat. The processes have significant influence on tastiness of meat, because of increasing of free and soluble amino acids and short peptides.

The aim of work was trying to increase the tenderness of beef meat using microbiological enzymatic preparations obtained from culture of Yarrowia lipolytica PII6a yeasts. Two enzymatic preparations, coming from Yarrowia lipolytica yeasts but rising on selective substratum, were tested. The first one (YI.O.) was obtained after the centrifuging of cultivation of yeasts that increased in the substratum where carbon came from sunflower oil, in the second one (Yl.G) glucose was the source of carbon.

Enzymatic preparations Yl.O was more effective against beef meat proteins than Yl.G. Both yeast preparations had better proteolytic activities than pepsin. All enzyme preparations showed different activity against beef meat proteins in different acidity of environment. Lengthening of tenderization time of beef meat to 48 hours had a significant influence on muscle proteins degradation. Although the degradation was the most intensive during first twenty-four hours. The best result of tenderness of beef was obtained after using Yarrowia lypolityca enzymes with oil as a carbon source and pH of environment 5.8.

Key words: tenderness, beef, enzymatic preparations, enzymatic hydrolysis.

INTRODUCTION

Tenderness of meat qualifies the cooking quality of meat. This parameter results from the structure of meat that depends of many pre-slaughter elements (genotype, age, gender) and post-slaughter [12, 13]. It is a part of texture of meat and its measure is resistance during biting or granulating. The tenderness can be evaluated using sensory or instrumental analysis [15]. The tenderness of meat is connected with protein of muscular tissue, proportions and changes between them during post-slaughter processes of meat. This parameter mainly depends of myofibril and connective proteins and their degradation after proteases activity. According to the present accepted theories, the determining agents during creating the tenderness of meat are proteins and their endogenous transformations [7, 13]. Functional properties and increasing of water-absorbing creates as results of post mortem transformations of meat. The processes have significant influence on tastiness of meat, because of increasing of free and soluble amino acids and short peptides [2, 10].

Process of formation good quality of beef lasts even 10-14 days. It can be shorten by different techniques: electrical stimulation, special under slinging and loading of carcass, adding exogenous enzymes which increase the speed of protein proteolysis [1, 12, 13].

The aim of work was trying to increase the tenderness of beef meat using microbiological enzymes obtained from culture of Yarrowia lipolytica PII6a yeasts.

MATERIALS AND METHODS

Tests were carried out on beef meat, which was treated with enzyme tenderization. The enzymes preparations were the products of Yarrowia lipolytica yeasts originating from the collection of Department of Biotechnology and Food Microbiology of Agricultural University in Wroclaw and pepsin.

Yarrowia lipolytica PII6a yeasts characterize of good proteolitic and lipolytic activity. The first activity is due to ability to synthesized proteases as well as in acid and basic environment. Whereas the lipolytic activity depends of presence the organic nitrogen sources in the environment: casein, peptone, soy protein and also inorganic, for example: ammonium sulfate [4, 16].

Two enzymatic preparations, coming from Yarrowia lipolytica yeasts but rising on selective substratum, were tested. The first one (YI.O.) was obtained after the centrifuging of cultivation of yeasts that increased in the substratum where carbon came from sunflower oil. In the second one (YI.G.), glucose was the source of carbon. The activity of the first enzyme preparation against hemoglobin was 1490 amu/cm³ and 14 unit/cm³ against casein. The activity of the second enzyme preparation against hemoglobin was 1230 unit/cm³ and 64 amu/cm³ against casein.

Each portion of meat (15 g) was treated with an enzyme preparation using the inundating method. The enzyme preparations came from liquids after the growth of Yarrowia lipolytica yeasts and Britton–Robinson’s buffer with the proper pH value in the amount of 30 cm³. Process of enzymatic hydrolysis of meat was carried out in 4°C during 4, 24 and 48 hours in 2 different acidities: pH 4.0 and 5.8. To compare, the degradation of proteins was also carried out using pepsin (Sigma) and the same parameters during researches were kept. The control group was meat treated with no enzyme but with only the buffer at pH 4.0 and 5.8. The level of degradation of proteins was determined by the amount of proteins [9], free amino acids [8, 14] and hydroxyproline [11] in post-hydrolysis solutions, as a difference between determination in proper and control probes. Moreover, the instrumental measurement of tenderness of beef meat, before and after the enzymatic hydrolysis that lasted 24 hours, was investigated (using Stevens QTS25). Statistical treatment of data was carried out using Statistica (ver. 6.0) and involved the single-element analysis and multi element analysis of variance.

RESULTS AND DISCUSSION

Results of chemical analysis and instrumental measurements of beef tenderness are shown at figures 1-4 and results of multi element analysis of variance are presented at table 1.

The amount of released products of enzymatic degradations increased during lengthening of degradation time (table 1). The biggest growth of analyzed parameters was obtained between 4 and 24 hour of hydrolysis of meat.

Table 1. Results of determining the content of products of enzymatic degradation of protein and shear force of beef. Multifactor analysis of variance

Factor Parameters	Enzymatic preparation			pH		Time[h]
	P	Yl.G	Yl.O	4.0	5.8	4	24	48
Protein [μg/g substate]	15236*)a	16141 b	19018 b	16891 a	17362 a	7879 a	18030 b	25472 c
Free amine groups [μg/g substrate]	154.83 a	162.71 a	277.70b	217.24 a	215.30 a	116.18a	218.67b	313.95c
Hydroxyproline [μg/g substrate]	7.88 a	11.77 b	12.21 b	9.63 a	10.46 a	7.78 a	9.91 b	12.45 c
Shear force [N/cm2]	61617 b	58711 b	48656 a	53849 a	56424 b	-	-	-

*) Means appearing in the same line and designated by identical letters indicate no statistically significant differences at a trust level of α≤ 0.05 (n=12).

Statistical treatment of protein measurements proved that there was no significant difference in the amount of proteins in solutions after 4 hours of enduring process, as well as when pepsin, in both pH values, and Yl.G in pH=5.8 was tested. Using Yl.O preparations caused the significantly bigger growth of released protein. The amount of proteins in solutions after a 24 hour degradation formed between 6.83 mg/g of substrate after treated by pepsin in pH 5.8 to 19.49 mg/g after the enzymatic degradation of meat using Yl.O in pH 5.8. Prolonged time of degradation had a significant influence on the level of proteins in solutions and the maximum proteins were found after treating the meat with Yl.O in pH 5.8 and it obtained 31.69 mg/g of substrate (fig. 1).

Figure 1. Content of protein in the solutions after enzymatic degradation of beef. One-way analysis of variance

Comparison of glycine amounts in different periods of time showed that the most intensive process of degradation was between 24 and 48 hour of lasting process at both pH values, when YL.O was the tendering factor. In this case, the bigger amounts were determined at lower pH of environment (pH=5.8). Testing the influence of Yl.G in different acidity on the efficiency of protein degradation (measured using both Lowry’s and freed amino groups method) demonstrated the opposite dependence and similar to pepsin, this enzyme preparation was more efficient at pH=4.0. The effect of pepsin got much worse results in comparison to both microbiological preparations (fig.2).

Figure 2. Content of free amine groups in the solutions after enzymatic degradation of beef. One-way analysis of variance

The results of ability to degradation collagen are demonstrated at figure 3. It must be stated that pepsin freed much less hydroxyproline from the substrate (maximum 10.87 µg after 48 hours at pH=4.0) than enzymes originated from yeasts (15.14 µg at pH=4.0 – Yl.G and 16.66 µg at pH=5.8 -Yl.O).

Figure 3. Content hydroxyproline in the solutions after enzymatic degradation of beef. One-way analysis of variance

Figure 4. Tenderness of beef after enzymatic degradation. One-way analysis of variance

The proteolytic activity of each preparation performed different in another environment acidity. The researches done by Czajgucka et al. [3] showed the similar dependence with PII6a Yarrowia lipolytica yeasts. Among 7 strains of isolated from cheeses, proteases of Yarrowia lipolytica yeasts demonstrated the highest proteolytic activity. Czajgucka et al. [3] proved that the highest activity of this proteases is observed at pH 7.0- 7.5 and the higher acidity cause worse activity of enzymes. The self researches indicated that enzyme preparation obtained from after-culture fluid with oil as a carbon source, was more effective at pH=5.8 and lower acidity was better for Yl.G and pepsin. Although, it must be noticed that pH=5.8 is closer to the acidity of meat destined to processing.

Presented comparison of enzymes efficiency showed large diversification of enzymes and statistical analysis (table 1) proved that degradation of beef meat proceeded more intensive with Yl.O than Yl.G and the least intensive with pepsin. According to these results, the source of carbon had a significant influence on proteolytic activity of preparations. Those statements are acknowledged by earlier experiments carried out by Krasnowska et al. [5, 6]. The level of growth of protein and glycine in solutions after the hydrolysis of muscle proteins was always higher when oil was the source of carbon in yeast culture. The effect of pepsin action was appeared to be much worse than yeasts preparations, but the amount of glycine, freed during meat protein degradation, for pepsin and Yl.G was approximated (154.84 µg – pepsin and 162.71 µg- Yl.G). Tested enzymes were not specific to degradate collagen and acted only on non-helical endings of collagen and this may be the reason of such a little amounts of released hydroxyproline. However yeast enzymes degradated this protein more effective than pepsin.

Analysis of chemical measurements results, presented at table 1, acknowledged the significance influence of enzymatic degradation time of beef, but it didn’t show the influence of environment acidity on significance of the measured level of solids.

The measurements of tenderness of meat, expressed by shear force, acknowledged the good effect of tenderization after using yeast preparations. The best results, corresponded to the lowest values of cutting force, were obtained in the Yl.O variant (48.6·10³ N/cm²), worse after using Yl.G preparation (58.7·10³ N/cm²) and the worst tenderization was observed after treating the meat with pepsin (61.6·10³ N/cm²). Those results also proved the statistic significance in difference of beef meat tenderness after degradation in different acidity. Results of multifactor analysis of variance showed that better tenderness of meat was acquired at hydrolysis at pH 4.0 (tab.1).

Wołoszyn i Szołtysek [17] also carried out experiments connected with using enzymatic preparations originated from microorganisms to change the structure of meat tissue. Experiments were carried out on fish tissue and enzyme preparations obtained from Penicillium roqueforti fungus. The tenderization effect was succeeded.

CONCLUSIONS

1. Enzyme preparation Yl.O was more effective against beef meat proteins than Yl.G. Both yeast preparations had better proteolytic (also collgenolytic) activities than pepsin.

2. All enzyme preparations showed different activity against beef meat proteins in different acidity of environment.

3. Lengthening of tenderization time of beef to 48 hours had a significant influence on muscle proteins degradation. Although the degradation was the most intensive during first twenty-four hours.

4. The best result of tenderness of beef was obtained after using Yarrowia lypolityca enzymes with oil as a carbon source and pH of environment 5.8.

REFERENCES

1. Buckenhüskes H.J., 2000. Enzyme in meat industry. Fleischwirtschaft, 3, 29-33.

2. Cegielska-Radziejewska R., Kijowski J., Tomaszewska-Gras J., 2004. Mięso i przetwory drobiowe [Meat and chicken products]. Pr. zb. pod red. T. Grabowskiego i J. Kijowskiego, WNT, Warszawa, 54-97 [in Polish].

3. Czajgucka A., Chrzanowska J., Juszczyk P., Szołtysik M., Wojtatowicz M., 2003. Aktywnosc proteolityczna szczepów drożdży pochodzšcych z serów Rokpol [Proteolytic activity of yeast strains originating from Rokpol cheese]. Biotechnologia. 2(1-2), 73-81 [in Polish].

4. Gdula A., Chrzanowska J., Szołtysik M., Kieżel X., Wojtatowicz M., 2002. Factors affecting hydrolytic enzymem productions by Yarrowia lipolytica strains. Biotechnologia, 1(1-2), 81-88.

5. Krasnowska G., 2004. Ocena własciwosci proteolitycznych preparatów enzymatycznych uzyskanych z różnych szczepów drożdży Yarrowia lipolytica [Proteolytic qualities of enzymatic preparations obtained from various strains of yeast Yarrowia lipolytica]. Żywnosc. Technologia. Nauka. Jakosc, 1(38), 94-103 [in Polish].

6. Krasnowska G., Sobków B., Dabrowska M., Pelc M., 2005. Okreslenie przydatnosci wybranych preparatów enzymatycznych do poprawy jakosci mięsa wołowego [Determination of the usefulness of selected enzymatic preparations to improve the quality of beef]. Żywnosc. Technologia. Nauka. Jakosc, 2 (43), 73-83 [in Polish].

7. Koohmaraie M., 1996. Biochemical factors regulating the toughening and tenderization processes of meat. Meat Sci., 43, 193-201.

8. Kuchroo C.V., Ramilly I.P., Fox P.F., 1983. Assessment of proteolysis in cheese by reaction with trinitrobenzensulphonic acid. J. Food Technol., 7, 129-133.

9. Mejbaum-Katzenellenbogen W., Mochnacka I., 1968. Kurs praktyczny biochemii. Oznaczanie białka metodš Lowry’ego [Practical course in biochemistry. Protein determination with Lowrie’s method]. PWN, Warszawa [in Polish].

10. The influence of post-mortem ageing and roasting on the microstructure, texture and collagen solubility of bovine semitendinosus muscle. Meat Sci., 64, 191-198.

11. PN-ISO 3496:2000. Oznaczanie hydroksyproliny [Hydroxyproline in meat and meat products] [in Polish].

12. Połczyńska I., Górska I., 1997. Czynniki kształtujace produkcje i jakosc kulinarnego mięsa wołowego w Polsce [Factor determining production and quality of culinary beef meat in Poland]. Żywnosc. Technologia. Nauka. Jakosc, 1 (10), 8-20 [in Polish].

13. Pospiech E., Grzeœ B., Iwańska E., 2003. Kruchosc mięsa kulinarnego i możliwosci jej poubojowego kształtowania [Tenderness of culinary meat and possibility its slaughter forming]. Rocz. Inst. Przem. Mięs. i Tł., 60, 71-82 [in Polish].

14. Snyder S.L., Sobociński P.Z., 1975. An improved 2,4,6-trinitrobenzosulphonic amid metod for determination of amines. Analysis Biochem., 64, 285-288.

15. Surówka K., 2002. Tekstura żywnosci i metody jej badania [Food texture and methods of measurement]. Przem. Spoż., 10, 12-17 [in Polish].

16. Robak M., Wojtatowicz M., 2002. Wykorzystanie drożdży Yarrowia lipolytica w procesach biotechnologicznych [The application of Yarrowia lipolytica yeast in biotechnological processes]. XXXVIII Zjazd PTBioch., Wrocław, 238-239 [in Polish].

17. Wołoszyn J., Szołtysek K., 1988. Zastosowanie preparatu enzymatycznego grzybni Penicillium roqueforti do zmiękczania tkanki kalmara i fladry [The application of fungal enzymatic preparation from Penicillium roqueforti to degradation fish tissue]. Przem. Spoż., 7, 212-219 [in Polish].

 

Accepted for print: 17.11.2006

---

Grażyna Krasnowska
Department of Animal Products Technology and Quality Management,
Agricultural University of Wrocław, Poland
C. K. Norwida 25/27, 50-375 Wrocław, Poland
Phone: 071 3205227, fax. 071 3205140
email: grazyna@ozi.ar.wroc.pl

Ewa Rudownik
Department of Animal Products Technology and Quality Management,
Agricultural University of Wrocław, Poland
C. K. Norwida 25/27, 50-375 Wrocław, Poland

Andrzej Hyliński
Department of Animal Products Technology and Quality Management,
Agricultural University of Wrocław, Poland
C. K. Norwida 25/27, 50-375 Wrocław, Poland

Anna Nowak
Department of Animal Products Technology and Quality Management,
Agricultural University of Wrocław, Poland
C. K. Norwida 25/27, 50-375 Wrocław, Poland

---

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

---

Main  -  Issues  -  How to Submit  -  From the Publisher  -  Search  -  Subscription