Metabolic, proteomic and microbial changes postmortem and during beef aging.

The purpose of this review is to provide an overview of the current knowledge about proteomic and metabolic changes in beef, the microbiological alteration postmortem and during aging, and observe the influence on beef quality parameters, such as tenderness, taste and flavor. This review will also focus on the different aging types (wet- and dry-aging), the aging or postmortem time of beef and their effect on the proteome and metabolome of beef. The Ca2+ homeostasis and adenosine 5'-triphosphate breakdown are the main reactions in the pre-rigor phase. After rigor mortis, the enzymatic degradation of connective tissues and breakdown of energy metabolism dominate molecular changes in beef. Important metabolic processes leading to the formation of saccharides, nucleotides, organic acids (e.g. lactic acid), creatine and fatty acids are considered in this context as possible flavor precursors or formers of beef flavor and taste. Flavor precursors are substrates for lipid oxidation, Strecker degradation and Maillard reaction during cooking or roasting. The findings presented should serve as a basis for a better understanding of beef aging and its molecular effects and are intended to contribute to meeting the challenges of improving beef quality.

Annotated draft genome sequences of Mucor flavus KT1a and Helicostylum pulchrum KT1b strains isolated from dry-aged beef surface.

Mucor flavus KT1a and Helicostylum pulchrum KT1b were isolated and identified in our earlier study as the two dominant fungal species on dry-aged beef. In this study, we report their genome sequences and annotations.

Antibacterial mechanism of metabolites of Lactobacillus plantarum against Pseudomonas lundensis and their application in dry-aged beef preservation.

In this study, the antibacterial mechanism of metabolites of Lactobacillus plantarum SCB2505 (MLp SCB2505) against Pseudomonas lundensis (P. lundensis) SCB2605 was investigated, along with evaluation of their preservative effects on dry-aged beef. The results demonstrated the effective inhibition of MLp SCB2505 on the growth and biofilm synthesis of P. lundensis. The treatment with MLp SCB2505 led to the compromised membrane integrity, as evidenced by reduced intracellular ATP content, increased extracellular AKPase, K+ and protein content, as well as disrupted cell morphology. Further metabolomics analysis revealed that MLp SCB2505 interfered amino acid metabolism, nucleotide metabolism, cofactor and vitamin metabolism, lipid metabolism and respiratory chain in P. lundensis, ultimately leading to the interrupted life activities and even death of the bacteria. Besides, MLp SCB2505 could effectively inhibit the growth of Pseudomonas in dry-aged beef and delay spoilage. These findings propose the potential application of MLp SCB2505 as an antibacterial agent in meat products.

Isolation of Debaryomyces hansenii and selection of an optimal strain to improve the quality of low-grade beef rump (middle gluteal) during dry aging.

OBJECTIVE: The objective of this study was to evaluate the effect of Debaryomyces hansenii isolated from dry-aged beef on the tenderness and flavor attributes of low-grade beef during dry aging. METHODS: Five D. hansenii strains were isolated from dry-aged beef samples. The rump of low-grade beef was inoculated with individual D. hansenii isolates and subjected to dry aging for 4 weeks at 5°C and 75% relative humidity. Microbial contamination levels, meat quality attributes, and flavor attributes in the dry-aged beef were measured. RESULTS: Of the five isolates, the shear force of dry-aged beef inoculated with SMFM201812-3 and SMFM201905-5 was lower than that of control samples. Meanwhile, all five isolates increased the total free amino acid, glutamic acid, serine, glycine, alanine, and leucine contents in dry-aged beef. In particular, the total fatty acid, palmitic acid, and oleic acid contents in samples inoculated with D. hansenii SMFM201905-5 were higher than those in control samples. CONCLUSION: These results indicate that D. hansenii SMFM201905-5 might be used to improve the quality of beef during dry aging.

Microbial, Physicochemical Profile and Sensory Perception of Dry-Aged Beef Quality: A Preliminary Portuguese Contribution to the Validation of the Dry Aging Process.

Beef dry-aging consists of a selection of unpackaged prime cuts placed in a controlled environment cold room for several weeks. The goals are to concentrate flavors like nutty and beefy and to improve tenderness. The aim of this study was to verify the microbiological and physicochemical behavior and sensory perception of meat during a sample process example of meat dry-aging. Twelve beef loins were selected for 90 days of dry aging and placed in a cold room with average temperature, relative humidity and forced air 3.2 ± 0.7 °C, 60.7 ± 4.2% and 0.5-2 m/s, respectively. Samples of crust and lean meat were collected on days 1, 14, 21, 35, 60 and 90 of the dry aging process for microbiological, physicochemical (pH, aw, color L*a*b*) and sensory analysis. During drying, no pathogenic bacteria were detected, and the average counts were higher for the crust. The average values for water activity (aw) and pH were 0.98 ± 0.02 and 5.77 ± 0.015, respectively. A slight decrease in aw and an increase in pH were observed over the aging period (p < 0.05). The lower microbial counts on the lean meat and the overall assessment of freshness confirm the importance of good manufacturing and storage practices during dry aging.

Direct Rub Inoculation of Fungal Flora Changes Fatty Acid Composition and Volatile Flavors in Dry-Aged Beef: A Preliminary Study.

Here, we established a method to produce dry-aged beef (DAB) by rub inoculation with fungal flora on the prepared DAB surface. Portions of Holstein steers' rumps were prepared by direct rub inoculation of fungal flora or without treatment (conventional DAB) and dry-aged for 26 days in an aging room at 2.9 °C and 90% relative humidity. We compared the fungal covering and meat quality, including fatty acid composition and volatile aromatic compounds, of fungal-inoculated DAB with those of the conventional DAB. The fungal-inoculated DAB was almost entirely covered with white mold, in contrast to the conventional DAB. Moreover, the proportion of oleic acid and the concentration of nine volatile compounds significantly increased in the raw meat of fungal-inoculated DAB compared with those in the conventional DAB (p < 0.05). These results suggested that direct rub inoculation of fungal flora from prepared DAB may accelerate DAB production and efficiently enhance the "melt-in-the-mouth" feeling and flavors of DAB.

Effects of Aging Methods and Periods on Quality Characteristics of Beef.

The objective of this study was to determine effects of aging methods (wet-aged, dry-aged, and packaged dry-aged) during 60 d on quality traits and microbial characteristics of beef. Wet-aged beef was packed by vacuum packaging and stored in a 4°C refrigerator. Dry-aged beef was used without packaging. Packaged dry-aged beef was packaged in commercial bags. Dry-aged and packaged dry-aged samples were stored in a meat ager at 2°C-4°C with 85%-90% relative humidity. Meat color, crust thickness, aging loss, cooking loss, Warner-Bratzler shear force (WBSF), texture profile analysis, Torrymeter, meat pH, water activity, volatile basic nitrogen (VBN), thiobarbituric acid reactant substances (TBARS), and microbial analysis were measured or performed every 15 d until 60 d of aging time. Meat color changed significantly with increasing aging time. Differences in meat color among aging methods were observed. Aging losses of dry-aged and packaged dry-aged samples were higher than those of wet-aged samples. Wet-aged beef showed higher cooking loss, but lower WBSF than dry-aged and packaged dry-aged beef. VBN and TBARS showed an increasing tendency with increasing aging time. Differences of VBN and TBARS among aging methods were found. Regarding microbial analysis, counts of yeasts and molds were different among aging methods at the initial aging time. Packaged dry-aged and dry-aged beef showed similar values or tendency. Significant changes occurred during aging in all aging methods. Packaged dry aging and dry aging could result in similar quality traits and microbial characteristics of beef.

Quality Properties of Bulgogi Sauce with Crust Derived from Dry-Aged Beef Loin.

This study aimed to determine the physicochemical properties (proximate composition, color, pH, salinity, water holding capacity (WHC), curing yield, and shear force) and sensory properties (electric nose and sensory evaluation) of Bulgogi sauce with added crust derived from dry-aged beef loin. Increasing the amount of crust in the Bulgogi sauce tended to increase the protein content, fat content, and pH. Uncooked Bulgogi also tended to have elevated fat content, ash content, pH, and shear force. Increasing the crust content tended to decrease the water content, lightness, redness, and yellowness of Bulgogi sauce. The yellowness of uncooked Bulgogi with 6%-12% crust in sauce was significantly lower than that of the control (no crust) and the sample with 3% crust in sauce (p<0.05). The redness of the cooked control Bulgogi was significantly lower than that of the samples with crust in sauce (p<0.05). The WHC of uncooked Bulgogi with 6%-12% crust in sauce was significantly higher than that of the control and the sample with 3% crust in sauce (p<0.05). The flavor, texture, and overall acceptability of the Bulgogi with 9% crust in sauce were significantly higher than those of the control (p<0.05). These findings showed that the crust did not degrade the physicochemical properties of Bulgogi sauce and meat. The sensory characteristics of Bulgogi marinated with 9% crust in sauce were rated the best as persensory evaluation. Therefore, crust is a suitable flavor enhancer for Bulgogi sauce, and a 9% addition amount is optimal in terms of quality.

Evaluation of Probiotic Characteristics of Newly Isolated Lactic Acid Bacteria from Dry-Aged Hanwoo Beef.

Dry aging is a traditional method that improves meat quality, and diverse microbial communities are changed during the process. Lactic acid bacteria (LAB) are widely present in fermented foods and has many beneficial effects, such as immune enhancement and maintenance of intestinal homeostasis. In this study, we conducted metagenomic analysis to evaluate the changes in the microbial composition of dry-aged beef. We found that lactic acid bacterial strains were abundant in dry-aged beef including Lactobacillus sakei and Enterococcus faecalis. We investigated their abilities in acid and bile tolerance, adhesion to the host, antibiotic resistance, and antimicrobial activity as potential probiotics, confirming that L. sakei and E. faecalis strains had remarkable capability as probiotics. The isolates from dry-aged beef showed at least 70% survival under acidic conditions in addition to an increase in the survival level under bile conditions. Antibiotic susceptibility and antibacterial activity assays further verified their effectiveness in inhibiting all pathogenic bacteria tested, and most of them had low resistance to antibiotics. Finally, we used the Caenorhabditis elegans model to confirm their life extension and influence on host resistance. In the model system, 12D26 and 20D48 strains had great abilities to extend the nematode lifespan and to improve host resistance, respectively. These results suggest the potential use of newly isolated LAB strains from dry-aged beef as probiotic candidates for production of fermented meat.

Analysis of aging type- and aging time-related changes in the polar fraction of metabolome of beef by 1H NMR spectroscopy.

The tenderness and taste of beef is improved by either dry- or wet-aging or a combination of both. The objective was to develop a validated method for detecting differences in the polar fraction of metabolome in dry-aged and wet-aged beef over the aging time and quantifying the metabolites of interest by 1H NMR spectroscopy using beef. Sixty strip loin (M. longissimus dorsi) samples aged in different ways (wet-aging vs. dry-aging) and aging times (0, 7, 14, 21, 28 days) were analyzed. The aging type could be defined by linear discriminant analysis with an accuracy of 95%. Ten (lactic acid, alanine, methionine, fumaric acid, inosine, inosine monophosphate, creatine, betaine, carnosine and hypoxanthine) out of eighteen metabolites differ significantly (p < 0.05) in content depending on the aging type. Fifteen metabolites in dry-aged and ten in wet-aged beef correlate with the aging time (r > 0.7, <-0.7), which shows significant aging time-related effects on the polar fraction of metabolome.

The effects of time and relative humidity on dry-aged beef: Traditional versus special bag.

The objective of this study was to evaluate the effects of relative humidity (RH) and different dry aging methods on the quality of beef. Sixteen loins, from eight carcasses, were used in this experiment. Each pair of loin was cut into eight sections with equal size, which were evenly assigned to eight treatments, by the combination of two dry aging methods (traditional and highly moisture-permeable special bag), two relative humidity (65 and 85% RH) and two aging times (21 and 42 days). At 85% RH, neither special bag nor the traditional dry aging methods were viable, since samples presented high microbiological counts, mucus and bad odor. At 65% RH, Enterobacteriaceae and lactic acid bacteria were not detected in any treatment. The highest aerobic plate count and psychrotrophic count were observed in the samples of the traditional dry-aged process whereas the special bag showed the greatest mold and yeast count. Regarding dry aging in special bag, there was a reduction in the weight loss (P < 0.05) and no change in the physical-chemical characteristics (P > 0.05) compared to traditional dry aging. The values of pH, moisture and Warner-Bratzler shear force were not affected (P > 0.05) by aging method and relative humidity. Thus, the results indicate that high RH should be avoided for both dry aging methods. Furthermore, the special bag dry aging can be considered an alternative to produce dry-aged beef, as it reduces weight losses even at conditions of lower relative humidity.

Dry-aged beef manufactured in Japan: Microbiota identification and their effects on product characteristics.

We aimed to determine the mold, yeast, and bacterial distributions in dry-aged beef (DAB) manufactured in Hokkaido, Japan, and to study their effects on meat quality compared to wet-aged beef (WAB). Two rump blocks from Holstein steer were dry- and wet-aged for 35 days at 2.9 °C and 90% RH. The psychrophilic molds Mucor flavus and Helicostylum pulchrum and other fungi (Penicillium sp. and Debaryomyces sp.) appeared on the crust of DAB, while lactic acid bacteria and coliforms were suppressed in the inner part of the meat. The composition of C16:0, C18:0, and C18:1 fatty acids did not differ between DAB and WAB, while more C17:0 fatty acids were detected in DAB. Dry aging suppressed acids and increased the production of various aroma compounds with mushroom-like, nutty, and other pleasant flavors. The meat quality and free amino acid (FAA) contents of DAB and WAB did not differ significantly. In this study, we identified major molds on DAB, which might contribute to an increase in aroma. Keywords: dry-aged beef; Mucor flavus; Helicostylum pulchrum; psychrophilic mold; meat quality; volatile aroma compounds.

Effects of freezing and thawing on microbiological and physical-chemical properties of dry-aged beef.

This study evaluated the effects of freezing, prior to and after dry aging, on the microbiological and physical-chemical quality of beef. Strip loins (n = 24) from 12 carcasses were assigned to four treatments: non-frozen dry aging (Dry); dry aging, steak fabrication, freezing and slow thawing (Dry + ST); freezing, fast thawing (FT; 20 °C/15 h) and dry aging (FT + Dry); freezing, slow thawing (ST; 4 °C/48 h) and dry aging (ST + Dry). Freezing conditions were - 20 °C/28 days and dry aging conditions were 2 °C/70% relative humidity, for 28 days. Freezing prior to dry aging did not affect the microbial counts compared to Dry. However, FT + Dry and ST + Dry increased (16%) total process loss (P < .05) compared to Dry and Dry+ST. Moreover, freezing changed volatile compounds profile. Thus, freezing prior to dry aging was not a feasible process due to increased process loss, while freezing after dry aging was considered a viable alternative to preserve the steaks without compromising beef physical-chemical traits.

High-pressure processing of usually discarded dry aged beef trimmings for subsequent processing.

In this study, we investigated if the usually discarded trimmings from dry aged beef can be incorporated into raw fermented sausages as a substitute for fresh beef without altering any major characteristics. Dry aged trimmings were subjected to high-pressure processing (600 MPa, 3 min hold) to reduce the bacterial load, achieving a 3-log reduction. HPP-treated dry aged beef trimmings were then incorporated into raw fermented sausages (60% pork and 40% beef). Beef was substituted with trimmings in different concentrations (7.5, 12.5, 25, 50, 100%). Due to the substitution, the water content of the sausages was reduced depending on the amount of beef substituted. Consequently, the sausages with substituted beef, for example, 50 and 100%, achieved the same water content after 5.4 and 3.7 days, respectively, than control sausage at day 9. However, the substitution (100%) affected the fat content, which contributes to significant differences (p < .05) in firmness during ripening.

Role of moisture evaporation in the taste attributes of dry- and wet-aged beef determined by chemical and electronic tongue analyses.

The role of moisture evaporation in the taste attributes of dry- and wet-aged beef was determined in this study. A total of 30 striploins (longissimus lumborum) were dry or wet aged for 28 days and analyzed for moisture content, taste-active compounds [free amino acids (FAAs), inosine 5'-monophophate (IMP), and reducing sugars], and taste attributes by an electronic tongue. After the completion of aging process, higher amounts of FAAs and reducing sugars were found in dry-aged beef (P < .05) in negative correlations with moisture content (r2 = -0.9 and - 0.9, respectively), which were not detected in wet-aged beef. However, the different taste attributes of dry- and wet-aged beef were observed by the electronic tongue from day 14, whereas their moisture content was significantly different only at day 28. Consequently, although the moisture evaporation during dry aging process contributed to the increased flavor of dry-aged beef, there are other factors affecting flavor development including microbial activity on the surface crust.

Changes in microbial composition on the crust by different air flow velocities and their effect on sensory properties of dry-aged beef.

Beef rumps (middle gluteal) were dry aged for 28 days using different air flow velocities of 0, 2.5, and 5 m/s (DA0, DA2.5, and DA5, respectively). The microbial composition, physicochemical traits (moisture, pH, and shear force), flavor compounds (inosine 5'-monophosphate, reducing sugar, free amino acid, and free fatty acid), and electronic tongue profile were analyzed at day 0, 14, and 28. No molds or yeasts were detected until day 14. On day 28, Pilaira anomala was found to be the most abundant in DA0, whereas DA2.5 and DA5 showed increased composition of Debaryomyces hansenii. With that, the significant changes in physicochemical traits and flavor compounds occurred. In addition, the pattern of flavor compounds and taste attributes from DA0, which had different mold and yeast compositions, were discriminable from DA2.5 and DA5. Therefore, our results suggest that air flow can affect microbial composition on the crust, possibly resulting in different sensory properties of dry-aged beef.

Storage Stability of Vacuum-packaged Dry-aged Beef during Refrigeration at 4°C.

Although the production of dry-aged beef has been increasing, most purveyors are unaware of the changes in quality that ensue after completion of the aging period and do not adhere to specific guidelines for its packaging and storage. The objective of this study was to investigate the storage stability of vacuum-packaged dry-aged beef based on changes in microbial, physicochemical, and sensory properties during refrigeration at 4°C for 21 d. The total aerobic bacterial count exceeded 6 Log CFU/g at approximately day 11 and significantly increased after day 14. Freshness indicators such as pH and volatile basic nitrogen content were acceptable until day 14 and 21, respectively. Based on the evaluation of overall sensory acceptability, the dry-aged beef was acceptable until 14 d without any sensory deterioration. Therefore, vacuum-packaged dry-aged beef could be stored for 11 d at 4°C without any adverse effect on its microbial and sensory quality.

Determination of Indicators for Dry Aged Beef Quality.

Previous studies on dry aged beef, which substantially increases the value of low-grade raw beef and non-preferred cuts, are currently limited to the observation of aged beef changes in laboratory settings or under particular aging conditions, whereas the factors influencing aging have so far been underexplored. Herein, we attempt to establish a technique for distinguishing between fresh and aged beef by observing changes in quality during beef aging. Specifically, we analyzed the effect of time on the quality of aged beef sourced from three Korean manufacturers and identified quality indicators that can be used to distinguish between fresh and aged beef, regardless of supplier. Storage/trimming/aging/cooking losses, moisture/fat/protein/collagen contents, and water holding capacity were tested as potential indicators, among other parameters. As a result, the quality of dry aged beef was shown to be supplier-dependent, which made the identification of factors for the above origin-independent discrimination difficult. Nevertheless, as storage loss, water holding capacity, and cooking loss significantly changed with dry aging time in all cases, these parameters were concluded to be potentially suited for discrimination purposes. The insights gained in this work may help promoting further research in this field and contribute to the development of a standard for consistent aged beef production.

Determination of Salable Shelf-life for Wrap-packaged Dry-aged Beef during Cold Storage.

We investigated microbial and quality changes in wrap-packaged dry-aged beef after completion of aging and subsequent storage in a refrigerator. After 28 days of dry aging (temperature, 4°C; RH, approximately 75%; air flow velocity, 2.5 m/s), sirloins were trimmed, wrap-packaged, and stored at 4°C for 7 days. Analyses of microbial growth, pH, volatile basic nitrogen (VBN), 2-thiobarbituric acid-reactive substance (TABRS), and instrumental color, myoglobin, and sensory evaluation were conducted on days 0, 3, 5, and 7. The results show that the number of total aerobic bacteria (TAB), yeast, and lactic acid bacteria increased with an increase in storage days, whereas no change in the growth of mold was observed during 7 days of storage. Based on the legal standard for TAB count, the estimated shelf-life of wrap-packaged dry-aged beef was predicted to be less than 12.2 days. However, the shelf-life should be less than 6.3 days, considering the result of sensory quality (odor, taste, and overall acceptance). No significant change in visible appearance was also observed during 7 days of storage. The results suggest that the present quality indicators for meat spoilage (pH, VBN, and TBARS) should be re-considered for dry-aged beef, as its characteristics are different from those of fresh and/or wet-aged beef.

Utilization of the Crust from Dry-aged Beef to Enhance Flavor of Beef Patties.

The crust that inevitably forms on dry-aged beef is usually trimmed and discarded before sale. The aim of this study was to explore methods for utilizing this crust in processed meat products. Four sirloins were dry-aged for 28 d at 4℃ (75% relative humidity). The crust obtained from the surface of the sirloins after completion of dry aging was lyophilized. Patties were prepared without added crust (control) or with 5% w/w crust, aerobically packaged, and stored at 4℃ for 7 d. Electronic nose analysis indicated that the volatile profile differed significantly between the patties with and without crust. Compared to the control patties, patties containing crust had higher flavor, tenderness, juiciness, and acceptability scores in a sensory panel evaluation (all p<0.05). In addition, patties with crust were less hard, gummy, and chewy than the control patties after 3 and 7 d of storage (all p<0.05). The number of total aerobic bacteria was higher in raw patties with crust than in the control patties during the storage (p<0.05). However, no pathogens were detected. 2-Thiobarbituric acid reactive substance values of patties containing crust were significantly lower than those of control patties after 2 and 6 d of storage (both p<0.05). Thus, crust from dry-aged beef can enhance the flavor by providing beefy and palatable flavor without a long period of dry aging.