Cowpea legumin preservative impacts on beef ribeye and implications on antibiotic resistant food borne pathogens

Wesam Dawam; Shimaa Edris; Ali Osman; Mai Elsheikh; Ahmed Hamad; Mahmoud Sitohy; Islam Sabeq

doi:10.1038/s41538-024-00337-z

. 2024 Nov 19;8:96. doi: 10.1038/s41538-024-00337-z

Cowpea legumin preservative impacts on beef ribeye and implications on antibiotic resistant food borne pathogens

Wesam Dawam ¹, Shimaa Edris ¹, Ali Osman ², Mai Elsheikh ¹, Ahmed Hamad ¹, Mahmoud Sitohy ², Islam Sabeq ^1,^✉

PMCID: PMC11577006 PMID: 39562784

Abstract

The current study assessed the minimum inhibitory concentration (MIC) of Cowpea-legumin (CPL) against antibiotic-resistant foodborne pathogens (FBP), the consequences on Longissimus thoracis et lumborum (LTL) quality and shelf-life, and the growth curves of inoculated FBPs. Fresh LTL-steaks were enriched with either 0.5 mg/g (CPL0.5) or 1 mg/g (CPL1) and evaluated over 15 chilling-days at 4 °C. Antibiotic-resistant Salmonella enterica and Escherichia coli were inhibited by 25 and 3.125 mg/mL, respectively, while antibiotic-susceptible FBPs and antibiotic-resistant Staphylococcus aureus were suppressed by 0.1 mg/mL CPL. CPL1-fortification produced fully tender LTL that was initially yellower/less red than the control, then turned brighter red with storage. CPL demonstrated promising dose-dependent antioxidant, and antibacterial activities against native spoilage and antibiotic-resistant/susceptible FBPs. CPL’s proteinaceous composition, besides sample size, might impact stability. Conclusively, CPL demonstrated promising preservative stability in fresh meat for a maximum of fifteen-days and represents a viable antimicrobial alternative in battle against antibiotic-resistance.

Subject terms: Antimicrobial resistance, Medical humanities, Applied microbiology, Pathogens, Health humanities

Introduction

Global consumption of beef proteins is expected to increase by 14% by 2030 compared to the base period average of 2018-2020, while beef protein availability is expected to increase by 5.9% by 2030¹. Over the last few decades, population growth and urbanization have led to a rapid increase in the diverse types of waste, including solid organic waste (including protein) generated in people’s daily lives and industrial production. Hence by 2050, about 340 million tons of waste will accumulate, compared to 249 million tons in 2030, representing a 36.5% relative increase².

Meat spoilage (MS) is a sophisticated process of microbial biodiversity that involves numerous distinct microbial interactions. The health of people is negatively impacted by MS, including the meat products waste along with significant losses in production, storage, transportation, and marketing³. Besides microbial spoilage, lipid and protein oxidation is the second most significant factor in meat deterioration in meat products. Meat palatability and other organoleptic qualities are generally affected with lipid and protein oxidation, and also diminish nutritional value, intrinsic and extrinsic quality, and other issues⁴. Controlling MS is primarily dependent on the development of novel technologies and tactics for the preservation of meat products, both of which rely on delaying the establishment and/or proliferation of natural flora. Among these approaches is the use of synthetic antioxidants and antimicrobials (e.g., nitrites, sulfites, benzoic acid, and sorbic acid) to extend the shelf life of meat and meat products and prevent meat spoilage caused by microbial, lipid, and protein oxidation⁵. Synthetic antioxidants and/or antimicrobials are recognized globally in commerce and, when used following the relevant regulations and the allowed dosage, are safe for consumers^6,7. Meat enterprises have been driven to replace synthetic antioxidants and antimicrobials in meat systems with plant-derived antimicrobial agents due to the public’s belief that natural compounds are generally safer and healthier⁸.

Antimicrobial resistance is a worldwide public health problem affecting humans and animals. Antimicrobial-resistant bacteria (AMR) cause around 2.8 million human diseases and 700,000 deaths yearly. This figure might climb to 10 million by 2050 if AMR is not effectively addressed⁹. Antimicrobial misuse is the leading driver of resistant bacteria growth in the food and environment, particularly in low- and middle-income nations. AMR-bacteria transmission from animals and the environment via food may result in the spread of resistance genes among bacterial species as well as between animals, humans, and the environment^10,11.

Furthermore, there has been an increase in antimicrobial-resistant (AMR) bacteria, including resistance to antimicrobials not licensed for use in veterinary medicine, which has been detected in meat products¹². These issues have prompted various food safety authorities to fight antimicrobial resistance to achieve optimal human health and well-being while also being internally related to animal health and the environment (One Health) because humans and animals are both affected by the same bacteria and are treated with the same antimicrobials¹³. Concerns regarding AMR bacteria, in addition to microbiological hazards, are among the major reasons food safety must be ensured. Natural antimicrobial approaches have been experimentally tested, such as using bacteriophages, antimicrobial peptides and proteins and phytobiotics, such as essential oils or propolis ethanol extracts^14,15.

Leguminosae seeds are high in biological value protein, carbohydrates, vitamins, and minerals and have been demonstrated to be natural sources of antioxidants, anti-inflammatory, and antibacterial substances¹⁶. Legume phytochemicals—which include proteins—have sparked a new line of investigations in the food sector, academia, and pharmaceutical enterprises to develop a new class of potentially valuable natural antibacterial and/or preservative components and extracts that could be used in innovative, clean-labeled products to address the threat of antibiotic resistance to public health^16,17. Additionally, plant-based ingredients blending into traditional meat products is the meat processing industry’s main priority in minimizing the negative effects on the environment, public health, and animal welfare¹⁸. Cowpea seed proteins, like soybean seed proteins, predominantly comprise globulins, which account for more than 51% of total protein composition, while albumins account for about 45%¹⁹. Previous research found that fortifying minced beef with chickpea legumin²⁰ and cowpea 7S & 11S globulin^14,21 had promising antibacterial benefits.

Additional investigations on legumes will be necessary to broaden the comprehension of multiple important but still unresolved topics, including the main antimicrobial compounds, mechanisms of action, microbial inactivation kinetics produced in novel matrices, MIC and MBC, antimicrobial activity stability, and formulation assays using real food matrices¹⁶. Another critical concern is their impact on the technological characteristics of food during shelf-life.

The current study attempts to determine whether increased Cowpea-legumin (CPL) levels can extend beef shelf-life beyond previous research findings, or whether its antibacterial and antioxidant stability is influenced by its proteinaceous composition and decreases over time. Thus, the goals of the current study included determining the minimum antibacterial concentration of CPL against antibiotic-resistant/susceptible food-borne pathogens and assessing their influence on shelf-life, antioxidant stability, quality attributes, and growth curves of artificially inoculated food-borne pathogens in beef longissimus steaks.

Results

Figure 1 depicts the minimal inhibitory concentration and biocidal activity of cowpea-legumin (CPL) against antibiotic-susceptible and antibiotic-resistant FBPs. In a turbidimetric resazurin-based microdilution method, CPL at 0.098 mg/mL killed all antibiotic-susceptible (ST72) and antibiotic-resistant Staphylococcus aureus (ST62). Similar biocidal effects were observed in antibiotic-susceptible Salmonella and E. Coli; however, suppression of growth or death was observed in resistant E. coli (EC55) and Salmonella (N7) at higher CPL dosages of 6.5 and 50 mg/mL, respectively (Fig. 1).

Fig. 1 — The isolates were classified as multidrug resistant (N7, EC55, and ST62) or non-resistant (EC39, N9, and ST72). All antibiotic-susceptible organisms and antibiotic-resistant *Staphylococcus aureus* were entirely eradicated by 0.098 mg/mL *CPL*; however, antibiotic-resistant *Salmonella enterica* and E. *coli* required higher biocidal doses of 50 and 6.25 mg/mL, respectively.

Over 15 chilling days, Table 1 illustrates the impact of the CPL pretreatment on various physicochemical and oxidative stability parameters of LTL-steaks compared to the control. According to statistics, there was a substantial difference in the estimated physicochemical qualities of LTL-steaks due to the interaction between pretreatment with CPL and the storage period. WHC and drip loss among all estimated physicochemical qualities of LTL-steaks did not vary due to the interaction between the CPL pretreatment and chilling duration (Table 1). Meanwhile, at various monitoring points across each group, the length of chilling duration significantly impacted all estimated physicochemical characteristics of LTL-steaks. Similarly, except for WHC, redness (a*), and Chroma, all calculated physicochemical properties of LTL-steaks were significantly altered by CPL pretreatment. In contrast to the ascending trend of the control LTL-steaks pH, which reached the spoiling point 9-days post-treatment, CPL pretreatment demonstrated an up-and-down pattern, attaining poor pH quality at the 15^th-day post-treatment (p < 0.05) (Table 1).

Table 1.

Shows the impact of the cowpea-legumin pretreatment on various physicochemical and oxidative parameters of Musculus longissimus thoracis et lumborum (LTL)-steaks compared to the control over 15 chilling days

Parameters	Groups	Chilling points (day)						SEM¹	P values
Parameters	Groups	1	3	6	9	12	15	SEM¹	Time	CPL¹	CPL *Time
pH	Control	6.05^Bd2	6.21^Cd	6.38^d	7.1^Ac	7.97^Ab	8.5^Aa	0.411	<0.001
	CPL1¹	6.22^Ae	6.33^Ac	6.16^f	6.38^Bb	6.29^Bd	6.76^Ba	0.086	<0.001
	CPL0.5¹	6.22^Ad	6.28^Bc	6.10^e	6.35^Bb	6.27^Bc	6.57^Ca2	0.064	<0.001
	SEM	0.004	0.006	0.095	0.018	0.004	0.007
	p value	<0.001	<0.001	0.452	<0.001	<0.001	<0.001		<0.001	<0.001	<0.001
WHC (%)¹	Control	93.17^ab2	88.10^b	89.08^abB	91.06^Aab	80.50^c	94.63^a2	1.167	<0.001
	CPL1	91.94	89.66	93.82^A	89.21^B	85.98	92.49	0.934	0.462
	SEM	0.599	0.550	0.975	0.369	1.749	2.061
	p value	0.22	0.218	0.027	0.046	0.136	0.274		<0.001	0.307	0.162
Purge loss (%)	Control	2.54^b	2.26^bB	4.15^ab	4.24^ab	5.67^ab	7.58^a	0.923	0.016
	CPL1	2.89^d	5.93^bcA	5.57^c	5.93^bc	9.27^a	8.09^ab	0.481	<0.001
	SEM	0.736	0.491	0.686	0.613	0.986	0.698
	p value	0.748	0.002	0.198	0.110	0.087	0.714		<0.001	0.010	0.412
Cooking loss (%)	Control	40.87	41.34	40.05	38.22	36.63	46.04	1.315	0.188
	CPL1	38.73^ab	41.49^a	40.15^ab	37.16^b	39.24^ab	42.44^a	0.782	0.019
	SEM	0.616	1.091	0.499	0.532	0.912	2.993
	p value	0.125	0.942	0.915	0.260	0.167	0.554		<0.001	0.001	<0.001
WBSF¹	Control	8.51^aA2	7.64^bA	9.12^aA	6.38^cA	8.55^aA	6.33^cA	0.483	<0.001
	CPL1	4.78^bcB	4.62^cB	4.36^cdB	4.08^dB	5.14^abB	5.37^aB	0.195	<0.001
	SEM	0.109	0.205	0.149	0.110	0.155	0.218
	p value	<0.001	<0.001	<0.001	<0.001	<0.001	0.016		<0.001	<0.001	<0.001
L*¹	Control	40.4^aA	39.4^abB	40.4^a	38.6^bcA	39.0^bc	38.1^cB	0.381	<0.001
	CPL1	39.5^cB	41.6^bA	40.24^c	36.8^dB	40.1^c	44.3^aA	1.016	<0.001
	SEM	0.116	0.167	0.153	0.065	0.530	0.230
	p value	0.012	0.001	0.687	<0.001	0.247	<0.001		<0.001	<0.001	<0.001
a*¹	Control	17.2^aA2	17.2^aA	17^a	15.6^bB	15.4^bA	15.1^bB	0.404	<0.001
	CPL1	16.0^cB	14.5^dB	16.6^bc	17.2^abA	14.5^dB	17.3^aA2	0.519	<0.001
	SEM	0.139	0.112	0.162	0.211	0.185	0.095
	p value	0.003	<0.001	0.196	0.009	0.031	<0.001		<0.001	0.293	<0.001
b*¹	Control	2.5^cB2	2.3^cdB	4.1^a	3.3^b	2.0^dB	2.01^dB	0.345	<0.001
	CPL1	3.1^bA	3.3^bA	3.4^b	3.5^b	3.0^bA	5.2^aA2	0.324	<0.001
	SEM	0.148	0.079	0.183	0.123	0.122	0.035
	p value	0.041	0.002	0.072	0.423	0.004	<0.001		<0.001	<0.001	<0.001
Chroma¹	Control	17.4^aA	17.3^aA	17.5^a	15.9^bB	15.5^b	15.2^bB	0.424	<0.001
	CPL1	16.3^cB	14.8^dB	16.9^b	17.6^aA	14.8^d	18.1^aA	0.558	<0.001
	SEM	0.119	0.115	0.134	0.181	0.180	0.101
	p value	0.003	<0.001	0.062	0.006	0.060	<0.001		.006	0.966	<0.001
Hue (h˚)¹	Control	8.2^Bb	7.7^Bb	13.6^a	12.1^a	7.3^Bb	7.6^Bb	1.109	<0.001
	CPL1	11.1^Ab	12.9^Ab	11.7^b	11.5^b	11.6^Ab	16.6^Aa	0.842	<0.001
	SEM	0.550	0.281	0.700	0.566	0.463	0.060
	p value	0.023	<0.001	0.166	0.470	0.003	<0.001		0.001	<0.001	<0.001
MDA (nM/g)¹	Control	70.05^b2	72.20^b	79.52^abA	92.55^abA	92.80^abA	113.96^aA	7.506	0.024
	CPL1	70.10	69.10	57.68^B	55.17^B	50.54^B	55.37^B	7.703	0.497
	CPL0.5	69.7^b	70.28^b	68.23^AB	73.49^AB	71.31^AB	83.63^AB2	69.71	0.488
	SEM	6.99	10.0	4.92	5.53	5.94	7.83
	p values	0.999	0.977	0.054	0.019	0.007	0.008		0.149	0.000	0.031

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¹CPL Cowpea-legumin, CPL1 1 mg/g cowpea-legumin inclusion, CPL0.5 0.5 mg/g cowpea-legumin inclusion, WHC water holding capacity, WBSF Warner-Bratzler Shear Force, L* Lightness, a* redness, b* yellowness, Chroma color intensity, h˚ color saturation or hue angle, MDA Malondialdehyde, SEM standard error of the means.

²Different small letters within the row show significant changes across chilling times (P < 0.05), while different capital letters within the column indicate significant differences between treatments.

LTL-steaks pretreatment with CPL did not affect estimated WHC throughout the entire chilling period and always had similar values to control meat (p > 0.05), except for the sixth-day post-treatment, which had a greater WHC value than the control (p < 0.05) (Table 1). On the other hand, purge loss was impacted by the chilling storage interval in all groups, and an elevated loss pattern was associated with longer storage, peaking at the 15^th chilling-day, clearly in control. CPL-pretreatment displayed no effect on LTL-steaks purge loss (p > 0.05), which remained comparable to control across all chilling-points with the exception of the third chilling-day, when CPL-pretreated LTL-steaks had greater drip loss values (p < 0.05) (Table 1).

Compared to the control, CPL1 pretreatment did not affect LTL-steaks cooking loss (CL) over the full experiment period (p > 0.05). The chilling interval demonstrated an effect on CPL1-pretreated LTL-steaks CL, but not on control; the lowest and highest CL occurred on the ninth and fifteenth chilling days, respectively (Table 1). Pretreatment of LTL-steaks with CPL1 reduced WBSF values compared to controls, and extended chilling time had a significant effect on this feature in both groups (p > 0.05). Until the ninth day post-treatment, CPL1 displayed a declining trend in the WBSF value (4.08) of LTL-steaks, but thereafter it increased dramatically (p > 0.05) (Table 1).

Except at the 6- and 12-day post-treatment, CPL1 had a significant effect on LTL-steaks lightness (L*) compared to the control (p < 0.05). Furthermore, lengthy chilling storage significantly impacted this feature; whilst it decreased with longer storage of control LTL-steaks (40.4 at first day to 38.1 at 15 days), the treated LTL-steaks exhibited the highest L* on the final day post-treatment (p < 0.05). Throughout the entire storage period, pretreatment of LTL-steaks with CPL1 decreased redness (a*) and chroma compared to control (p < 0.05); however, on the 9-and 15-day post-treatment, it increased (p < 0.05). Additionally, longer storage significantly reduced the a* and chroma of the control LTL-steaks. In contrast, the treated LTL-steaks showed a roughly climbing trend, except for the 12- day post-treatment, which interestingly presented the lowest a* (14.5) and chroma (14.8) comparable to the 3- day (p < 0.05). Pretreatment of LTL-steaks with CPL1 resulted in increased yellowness (b*) and hue (h˚) compared to the control group (p < 0.05), with comparable results at the sixth- and ninth-days post-treatment (p > 0.05). Extended chilling storage also had a substantial impact on b* and h˚ in the compared groups; whereas b* and h˚ of the control group showed a rising trend up to their highest values at the sixth- and ninth-day post-treatment, they thereafter showed a significant decline until the conclusion of the storage period (p > 0.05). CPL1 kept LTL-steaks b* and h˚ uniform throughout the chilling storage experiment but reached their peak at the 15-day post-treatment (p > 0.05) (Table 1).

Oxidation indicators revealed that both CPL pretreatment levels, CPL1 and CPL0.5, greatly enhanced the LTL-steaks oxidative stability compared to the control, having a consistent effect throughout the whole storage period as opposed to the control’s noticeably rising curve. Also, the interaction between storage length and CPL pretreatment significantly affected oxidative stability (p < 0.05) (Table 1).

Table 2 illustrates the influence of the two CPL inclusion levels, CPL1 and CPL0.5, on different LTL-steaks bacteriological indices and shelf-life compared to control over 15 chilling-days. Statistically, all LTL-steaks bacteriological indices were affected by the Cowpea-legumin pretreatment levels, storage interval, and their interaction (p < 0.05). Both CPL pretreatment levels, CPL1 and CPL0.5, retarded all LTL-steaks bacteriological indices, APC, coliform, Lactic acid bacteria (LAB), and Staphylococcus count, of LTL-steaks from the first day compared to the control (p < 0.05). The CPL antimicrobial influence continued till the end of chilling storage at fifteen-day post-treatment (p < 0.05). Higher inhibition corresponded to higher CPL inclusion levels. Both CPL inclusion levels were able to retard APC below 6 logs CFU/g till the twelve-day post-treatment. The CPL antimicrobial effect was higher on Staphylococcus count than coliform, where higher inclusion levels retarded the Staphylococcus growth curve till the end of storage within the range of 2 to 2.6 log CFU/g. Also, significant differences were noticed between the six checkpoints of the growth curve of different estimated bacteriological indices of control and CPL-pretreated LTL-steak groups (p < 0.05). Also, the LAB growth curve was retarded by CPL inclusion levels till the fifteen-day post-treatment, with dose-dependent effects (p < 0.05).

Table 2.

Shows the influence of the two Cowpea-legumin (CPL) inclusion levels on different Musculus longissimus thoracis et lumborum (LTL)-steaks bacteriological indices and shelf-life compared to control over 15 chilling days

Parameter	Groups	Chilling points (day)						SEM¹	P values
Parameter	Groups	1	3	6	9	12	15	SEM¹	Time	CPL¹	CPL *Time
APC¹	Control	4.86^Ac2	5.15^Ac	5.27^Ac	7.09^Ab	7.62^Ab	8.29^Aa	0.599	<0.001
	CPL1¹	4.37^ABcd	3.13^Be	3.87^Bd	4.54^Bbc	5.12^Bb	6.31^Ba	0.445	<0.001
	CPL0.5¹	3.70^Bd	3.83^Bd	4.08^Bd	4.74^Bc	5.49^Bb	6.69^Ba2	0.473	<0.001
	SEM	0.129	0.168	0.073	0.149	0.082	0.138
	p value	0.014	0.001	<0.001	<0.001	<0.001	<0.001		<0.001	<0.001	0.007
Coliform	Control	4.22^Ad2	4.41^Ad	4.91^Ac	6.78^Ab	7.77^Aa	7.86^Aa	0.685	<0.001
	CPL1	2.99^Bc	3.01^Cc	2.69^Ccd	3.19^Bc	4.21^Cb	5.72^Ba	0.443	<0.001
	CPL0.5	2.99^Bc	3.41^Bb	3.14^Bbc	3.16^Bbc	5.35^Ba	5.65^Ba2	0.524	<0.001
	SEM	0.036	0.029	0.086	0.061	0.015	0.196
	p value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001		<0.001	<0.001	<0.001
LAB¹	Control	4.47^Ac2	5.26^Ab	5.64^Ab	7.85^Aa	7.54^Aa	7.57^Aa	0.589	<0.001
	CPL1	4.09^Cb	3.90^Bb	3.60^Bb	4.01^Cb	5.35^Ba	6.11^Ba	0.404	<0.001
	CPL0.5	4.16^Bbc	3.94^Bcd	3.69^Bd	4.45^Bb	5.57^Ba	5.74^Ba2	0.351	<0.001
	SEM	0.012	0.151	0.097	0.069	0.199	0.142
	p value	<0.001	0.001	<0.001	<0.001	0.001	0.002		<0.001	<0.001	<0.001
Staphylococcus	Control	3.67^d	3.17^Ad	3.58^Ad	4.57^Ac	5.31^Ab	6.34^Aa	0.494	<0.001
	CPL1	3.74^a	3.06^Ab	2.0^Bc	2.0^Cc	2.2C^c	2.0^Bc	0.297	<0.001
	CPL0.5	3.74^a	2.26^Bcd	2.0^Bd	2.56^Bc	3.24^Bb	2.0^Bd	0.291	<0.001
	SEM	0.051	0.079	0.017	0.069	0.157	0.007
	p value	0.741	0.001	<0.001	<0.001	<0.001	<0.001		<0.001	<0.001	<0.001

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¹CPL Cowpea-legumin, CPL1 1 mg/g cowpea-legumin inclusion. CPL0.5 0.5 mg/g cowpea-legumin inclusion, SEM standard error of the mean; APC aerobic plate count, LAB Lactic acid bacteria.

²Different small letters within the row show significant changes across chilling times (p < 0.05), while different capital letters within the column indicate significant differences between treatments.

Table 3 illustrates the antimicrobial influence of the two CPL inclusion levels over 15 days of chilling on experimentally infected LTL-steaks with Listeria monocytogens and Salmonella compared to control and Sodium nitrite (NaNO₂) pretreated LTL-steaks. Statistically, Listeria monocytogens and Salmonella growth curves were affected by the interaction of CPL inclusion levels and chilling storage length (p < 0.05). The CPL pretreatment levels, particularly CPL0.5, significantly inhibited Listeria monocytogens growth curves compared to control LTL-steaks from immediate inclusion at 1^st day to the end of chilling storage (p < 0.05). NaNO₂ also showed comparable inhibitory influence on most checking-days of Listeria monocytogens growth curves compared to control. Both CPL1 and CPL0.5, as well as NaNO₂, had an immediate growth retardation effect on Salmonella growth curves until fifteen days post-treatment, keeping the Salmonella peak growth count below 2.76, 3.19, and 2 logs CFU/g, respectively, compared to 3.75 logs CFU/g for the control group. Notably, NaNO₂ expressed a higher constant inhibitory influence than CPL.

Table 3.

Antimicrobial influence of the two Cowpea-legumin (CPL) inclusion levels over 15 days of chilling on experimentally infected Musculus longissimus thoracis et lumborum (LTL) steaks with Listeria monocytogens and Salmonella compared to control and Na nitrite pretreated LTL-steaks

Pathogen	Groups	Pathogen growth throughout 15 days						SEM¹	P value
Pathogen	Groups	1	3	6	9	12	15	SEM¹	Time	CPL¹	CPL *Time
Listeria monocytogens	Control	3.18^Ad2	3.54^Bd	4.31^Ac	4.53^Ac	5.06^Ab	6.80^Aa	0.525	<0.001
	CPL1¹	2.65^Bb	3.78^Ba	2.85^Cb	2.64^Cb	2.65^Cb	3.45^Ba	0.200	<0.001
	CPL0.5¹	3.0^ABcd	3.15^Cbc	3.85^Ba	3.23^Bbc	2.72 ^Cd	3.52^Bab	0.162	<0.001
	Nitrite	2.65^Bc	4.73^Aa	2.0^Dd	2.0^Dd	4.33^Bb	2.0 ^Cd2	0.512	<0.001
	SEM	0.100	0.063	0.045	0.054	0.080	0.131
	p value	0.025	<0.001	<0.001	<0.001	<0.001	<0.001		<0.001	<0.001	<0.001
Salmonella enterica	Control	3.1^Aab2	3.02^b	2.96^Ab	3.25^Aab	3.52^Aab	3.75^Aa	0.123	0.019
	CPL1	2.69^Ba	2.0 ^Bb	2.0^Bb	2.0^Bb	2.0^Bb	2.0^Bb	0.116	<0.001
	CPL0.5	2.98^ABb	2.0^Bc	2.0^Bc	2.0^Bc	3.2^Aab	3.4^Aa	0.281	<0.001
	Nitrite	2.67^B	2.0^B	2.0^B	2.0^B	2.0^B	2.0^B2	0.112
	SEM	0.079	0.015	0.015	0.007	0.077	0.120
	p value	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001		<0.001	<0.001	<0.001

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¹CPL cowpea-legumin, Control, CPL1 1 mg/g cowpea-legumin inclusion, CPL0.5 0.5 mg/g cowpea-legumin inclusion, SEM standard error of the mean, Nitrite, minced LTL blended with sodium nitrite at 150 PPM.

Discussion

The study’s objectives included determining the minimum antibacterial concentration of CPL against antibiotic-resistant/susceptible food-borne pathogens in vitro, as well as the effects of CPL on native spoilage flora, antioxidant stability, quality attributes, and growth curves of artificially inoculated food-borne pathogens in beef LTL-steaks. As evidenced by the resazurin turbidimetric microdilution experiment, 0.098 mg/mL CPL inhibited the growth of Staphylococcus aureus, which is both susceptible and resistant to antibiotics (ST72 and ST62). Antibiotic-susceptible Salmonella and E. coli were inhibited by the same minimum inhibitory concentration (MIC); however, antibiotic-resistant E. coli (EC55) and Salmonella (N7) were controlled by higher CPL MICs of 3.125 and 25 mg/mL, respectively. Although they employed a different method, the agar well diffusion test, earlier studies on legume proteins revealed that doses as low as 0.098 g/mL might suppress particular pathogens²⁰. Additionally, 0.5 and 1 microgram of 7S and 11S globulins from red kidney beans (Phaseolus vulgaris L.) could inhibit antibiotic-resistant K. pneumoniae and E. Coli, according to a previous study on antibiotic-resistant Enterobacterales²². On the other hand, larger concentrations of different legume proteins (500 mg/mL) were added to milk; nonetheless, the MIC was ascertained in vivo right away without having to arrange for additional in-vitro testing²³. Both current and previously published research support evidence that legume proteins, such as cowpea-legumin, have a promising antibacterial impact against MDR Gm-positive and Gm-negative bacteria and that they could be used as an alternative to routinely applied antibiotics to help combat antibiotic resistance²⁴.

The findings showed that the inclusion of CPL from the first day was linked to a higher pH_U than the control group (Table 1). This finding could be explained by the proteinaceous and /or alkaline nature of cowpea-legumin (CPL)²¹; the dose-dependently higher pH of the two legumin-fortified LTL-steaks than control groups could support this assumption. The CPL-pretreated LTL-steaks produced evident spoilage pH after 15 days in both levels, whereas control LTL-steaks reached borderline pH after 6 days and apparent spoilage at 9 days (Table 1). Time-dependently raised pH can be attributed to the generation of spoilage metabolites, which are invariably produced with extended storage times due to oxidative and microbiological damage²⁵. In this case, CPL was able to postpone these adverse interactions at every level.

The pH_U has been extensively employed as a potential meat quality predictor²⁶. Higher WHC is always associated with higher pH²⁷; however, in the present situation, there was no difference in WHC between the control and CPL-fortified groups, except for the sixth day, when the LTL-steak received CPL expressed higher WHC (Table 1). At this stage, the CPL addition produced this activity since the control group had a lower WHC but a higher pH.

Excessive purge loss from fresh meat correlates with losses in water, flavoring compounds, vitamins, and minerals, in addition to the monetary expenses associated with such products²⁸. Both compared LTL-steak groups generated ascending purge loss curves with longer storage time and generated similar loss values except on the third-day post-treatment. The CL values of the two compared LTL-steak groups exhibited comparable findings throughout the trial, in line with purge loss (Table 1). The CL values of the control LTL-steaks did not vary throughout the six monitoring-points, while the CPL1 inclusion showed a similar pattern to pH (Table 1). Previous research found that non-meat protein addition, such as leguminous Pea protein isolates, can increase the surface area of meat proteins that come into contact with water, improving water binding capacity and yield during cooking^18,29,30. Excluding minor details, the tiny quantity of CPL used in the current investigation may be attributed to equivalent results of WHC, drip loss, and cooking loss between control and CPL-pretreated LTL-steaks. Furthermore, it has been noted that the 7S and 11S denature at temperatures of around 70°C and 90°C, respectively, which is necessary to acquire or improve technological capabilities³¹. This temperature may not have been attained under the current study cooking conditions, preventing CPL from experiencing sufficient structural changes during cooking, restricting their ability to interact with other components and their emulsification potential³².

Meat tenderness is frequently recognized as among the most essential quality features influencing customer acceptance of fresh meat³³. The extent of structural myofibrillar protein integrity/degradation (proteolysis), the degree of sarcomere contraction or “sarcomere length,” and the composition and content of connective tissue are the key elements that determine meat tenderness³⁴. The occurrence of meat tenderness is connected to the postmortem endogenous proteolytic degradation of muscle proteins. Changes in intramuscular circumstances, however, such as a decrease in pH, an increase in ionic strength, a decrease in reducing power, and the accumulation of reactive oxygen and nitrogen species, trigger this process³⁵. Previous research has shown that the degree of meat tenderness and pH are correlated; as meat ages, high-pH meat tenderizes more quickly than low-pH meat. Here, however, the pH and WBSF outcomes demonstrate that the three proteolytic systems—cathepsin B, μ-calpain activities, and multicatalytic proteinase complex— perhaps were activated by CPL. Previously, suboptimal cathepsin B activity and intermediate μ-calpain activities were linked to intermediate pH_U (5.8 to 6.19)³⁶. Additionally, CPL may counteract small heat shock proteins linked to harder meat at intermediate pH_U, as observed in the control LTL-steaks³⁷. Higher pH ultimate values (>6.2) in CPL-pretreated LTL-steaks than in control (intermediate pH_U, 5.8 to 6.19) observed on the first day explain realistically that the addition of CPL provided a high pH (>6.2) environment in pretreated LTL-steaks that lasted until the twelve-day post-treatment, stimulating the interaction of the three components to promote tenderness process. This hypothesis³⁸ could help to explain the overall low WBSF scores in CPL-pretreated LTL-steaks (Table 1). According to a previous report, the present tenderization capacity of CPL inclusion is classified with intermediated tenderness. A steak is deemed “tender” if its WBSF value falls between 3.36 and 4.28, and “intermediate tender” if its value falls between 4.3 and 5.4 kgf³⁹.

In contrast to the clear dropping tendency in control LTL-steak’s L*, a* and chroma with longer storage duration, CPL1 inclusion dropped L*, a* and chroma until the twelve-day compared to the last checking-day post-treatment (15), with a barely increasing trend. Meanwhile, LTL-steaks b* increased with CPL1 inclusion and prolonged storage, except for the middle storage phase between the sixth- and ninth-days post-treatment, which did not differ from the control (Table 1). CPL1-pretreated LTL-steaks demonstrated less redness intensity than the control, defying the yellowness trend (Table 1). Prior studies revealed that the addition of plant protein, such as pea protein isolate, was associated with a dose-dependent increase in the b* value and a decrease in red products; nevertheless, there were differences in the L* values of meat products, ranging from low^3,40 to high³⁰. Here, CPL-pretreated LTL-steaks produced low, equivalent, and high L* values up until the end of storage (Table 1). The results of the most current study, which used soybean 11S globulin (SBG) on LTL-steaks, showed that most color parameters did not change from the control group, with the exception of yellowness and hue, which were higher in the LTL-steaks supplemented with SBG⁴¹. Previously, sensory analysis of minced beef supplemented with CPL demonstrated that the color was overall consistent during 4 °C storage²⁰. The CPL inclusion, on the other hand, reduced myoglobin oxidation in a dose-dependent manner as compared to the decreasing redness pattern of control minced beef, which was potentially explained by higher oxidation to metmyoglobin levels in the control samples²⁰. In shrimp, CPL spray could prevent oxidative rancidity and melanosis while keeping fresh color compared to control⁴².

Current oxidation results demonstrated that both CPL levels significantly and dose-dependently improved the oxidative stability of LTL-steaks compared to the control during the entire storage period (Table 1). Although different color evaluation methods were used²⁰, the fewer red LTL-steaks obtained here could be attributed to the greater CPL content used (1 mg/g). However, the observation that the CPL-fortified LTL-steaks remained red until day twelve post-treatment when compared to the interrupted control group indicates that the antioxidant activity of CPL is sufficiently strong to stop the conversion of oxymyoglobin to metmyoglobin⁴³ during this stage of the experiment. Previous research proposed two mechanisms for soybean protein antioxidant activity: protective and bioactive peptides. In the protective pathway, SBG binds noncovalently with highly oxidizable lipids and lipid-soluble nutrients such unsaturated fatty acids, enhancing oxidative stability and preventing oxidative degradation^44,45. Second, the bioactive peptides produced by hydrolysis of SBG and boosted by the release of amino acids are thought to be responsible for the bioactive peptides’ high capacity to scavenge free radicals, reduce power, inhibit lipid peroxidation, and chelate metal ions^46,47.

All LTL-steaks bacteriological indices were negatively influenced by CPL-pretreatment levels in a dose-dependent manner, with both low and elevated levels keeping APC below the spoilage level of 6 log CFU/g for 12 days (Table 2). Similarly, chickpea legumin supplementation (200 μg/mL) dramatically reduced APC, psychrotrophic count, and coliform counts in minced beef, improving the preservation period to 15 days²⁰. Further, CPL exhibited a higher antibacterial effect on Staphylococcus counts than coliform (Table 2). Osman et al.²⁰ also noticed that chickpea legumin supplementation had a higher inhibitory effect on Staphylococcus aureus (57%) than Gram-negative bacteria, E. coli (29%) and P. aeruginosa (51%) in minced beef. In terms of pH and microbiological assessments, CPL considerably increased the shelf-life of LTL-steaks at 4 °C from 6 days to 12 days. These results are consistent to previous findings of CPL on minced beef, where notable reduction in coliforms, psychrotrophs, and APC which remained below six log CFU/g even after 15 days of storage at 4 °C when treated with 50 and 100 µg/g of CPL²¹. The reasons for higher reduction activity in minced meat than in existing LTL-steaks may be due to the easily and uniformly dispersion of CPL powder when applied to minced meat rather than steak pieces weighing 60 g⁴⁸. Previous investigations on extending the shelf-life of meat products revealed that the antimicrobial activity of powdered plant is less effective when used as a marinade and on whole meat as opposed to minced meat. This is because meat has the ability to act as a buffer, neutralizing the acidic marinade and causing lipophilic acids to dissociate^48,49. Here, DW was added to enable even distribution of CPL on LTL-steaks with contact for half an hour, which appears to be ample time for meat to release buffering components, as demonstrated when meat pH is evaluated. Furthermore, the current CPL antimicrobial stability results were similar to earlier research on shrimp treated with 100 µg/ml and 200 µg/ml of CPL, which significantly reduced APC below 5.98–5.15 log CFU/g over 12 days, respectively⁴². The explanation for the higher reduction action in shrimp compared to the present steaks could be related to the 2 ± 0.5 °C chilling temperature. Controlling temperature and moisture is the foundation of practical strategies for preserving fresh meat⁵⁰. Furthermore, other leguminous globulins, such as soybean glycinin (11S) in LTL-steaks⁴¹ and minced meat, and chickpea legumin (11S) in minced meat, shown equivalent and superior antibacterial activity than existing CPL in steak^20,23. Previously, it was reported that cowpea seed protein globulins induced entire membrane degeneration, cell enlargement, vacuole development, and, finally, full lysis of cell components²¹. Similarly, swelling and disintegration of the bacterial cell membrane of both Gram-negative and Gram-positive bacteria have been reported as one of the antibacterial properties of soybean glycinin (11S) and chickpea legumin applied in beef and milk^20,23.

Previous research, rather than using food matrix, concentrated on the invitro antimicrobial assessment of isolated legumes protein and peptides, soybean glycinin (11S) and chickpea legumin (11S)^20,23, against food-borne pathogens such Staphylococcus aureus, E. coli, and P. aeruginosa on agar media. Here, however, the antibacterial impact and stability of the two CPL inclusion levels were estimated on experimentally contaminated LTL-steaks with Listeria monocytogens and Salmonella enterica subspecies enterica during 15 days of chilling versus control-negative beef and Na nitrite (NaNO₂) pretreated meat (Table 3). The risk of contaminated meat and meat products arises from the vast distribution and several survival strategies of Listeria monocytogenes in raw materials, processing facilities, and retail environments, including vacuum (VP) conditions^51,52. Listeria monocytogenes (LM) is a psychrotrophic bacterium that grows and adapts faster at 4 °C than at the greater chilling temperature, unlike mesophilic classified Salmonella⁵³. This explains the considerable LM increase in control beef during refrigerated storage. However, cowpea-legumin pretreatment, especially at high levels, significantly decreased Listeria monocytogens growth curves from the first hour of incorporation to the end of chilling storage. The LM maximum count in CPL-treated meat ranged from 3.45 to 3.52 log CFU/g, while the LM maximum count in control beef was 6.80 log CFU/g, displaying a three-log decline (Table 3). Prior studies on the safety of meat products have established that synthetic nitrates and NaNO₂ are crucial in reducing pathogens like Clostridium botulinum, Listeria spp., and Salmonella spp. They also provide a larger margin of safety and cannot be excluded from the hurdle technology during the process^54–56. Both pathogen counts, especially those of Salmonella, were lower at all measured chilling-points in positive control LTL-steaks containing NaNO₂ compared to control. For artificially inoculated pathogens in minced LTL-steaks, especially LM, neither natural CPL nor synthetic NaNO₂ exhibited a uniform dropping curve despite their antibacterial interactions being equivalent in most of the checkpoints assessed. Pathogenic bacteria compete for AMPs with organisms for nutritional and ecological demands, resulting in greater flow through the cell wall. Through their hydrophobic and hydrophilic properties, AMPs can usually target many sites on pathogen plasma membranes and intracellular components, but they have limited cytotoxicity to mammals like as humans⁵⁷. These advantages over existing antibiotics may explain the promising antibacterial influence of currently investigated CPL and their potential as medications in human infection and the meat sector compared to chemical preservatives.

The differences in shelf-lives observed between the current CPL and other studies conducted with either the same or different leguminous globulins provide insight into the factors that may affect the antibacterial efficacy and/or preservation stability of CPL when applied to fresh meat, as well as the most practical methods of application. The reason behind the limitation of CPL’s antimicrobial stability to only twelve chilling days, even with a higher pretreatment dose applied in the current study compared to previous studies, could be attributed to the native bacterial population and their defense mechanisms against natural antimicrobials, along with meat size, shape, pH, and temperature. Unfortunately, previous findings have shown that pathogenic bacteria, particularly resistant ones, can adapt to naturally occurring antimicrobials such peptides and AMP by developing pathways comparable to those of antimicrobial medicines. Modifications to the membrane or cell wall structure to obstruct one of the main AMP-based killing functions (i.e., to halt interaction with the negatively charged membrane), express resistance genes, sequester or inactivate AMP molecules, and remove them via efflux pumps or ABC transporters are some of these techniques⁵⁸. Probable allergy is another factor that restricts the use of CPL as a fresh mat preservative. Although peas are not a priority allergenic food, Pis s1 and Pis s2 have been proposed as very rare possible allergens derived from vicilin and convicilin^59,60.

In conclusion, CPL demonstrated promising antibacterial activity against LTL-steaks native flora, inoculated food-borne Gram-positive and -negative pathogens, and dose-dependent antioxidant activities that extended LTL-steaks freshness and improved tenderness by over twelve cooling days. CPL antibacterial efficacy and stability are controlled not only by dose and temperature, but also by matrix particle size, which has an impact on powder distribution and interaction. Moreover, the addition of CPL results in less red and yellow LTL-steaks in comparison to the control, but with extended storage times, the antioxidant capacity of CPL maintains color.

Methods

Experiment management and approval

Under the number BUFVM 10-06-2023, the Institutional Animal Care and Use Committee Research Ethics number (BUFVTM) of Benha University’s Faculty of Veterinary Medicine approved all the protocols used in this investigation. All related life science studies declarations are not applicable because the present study did not report results on humans or animals.

Cowpea 11S globulin (legumin) protein extraction

The 7S globulin (vicilin) and 11S globulin (legumin) protein subunits were harvested from the defatted powder of cowpea seeds (Vigna unguiculata (L.) Walp.) using the previously published procedures with minor changes²¹. Centrifugation at 5000 × g for 10 min was carried out to extract the solubilized contents of 10 g of defatted cowpea seed powder dissolved in 150 mL of buffer (0.03 mol/L tris HCl at pH 8.5, 0.4 M NaCl, 10 mM -mercaptoethanol, 1 mM EDTA, 0.02% (w/v) NaN3). A 45°C water bath was used to stir the solution for 1 h. It was necessary to precipitate the 7S and 11S globulins using ammonium sulfate (50–65% and 65–85%, respectively). To precipitate 11S globulins, 65–85% ammonium sulfate was used. The precipitate was disseminated and solubilized as described above before being dialyzed against the same buffer for 48 h to remove the salts.

The preparation of pathogens and inoculums

Two isolates of Salmonella enterica subspecies enterica (N7 from liver and N9 from minced meat), Escherichia coli (EC39, EC55, both from minced meat), and Staphylococcus aureus (ST62 from frozen minced meat and ST72v)—either multi-drug resistant (N7, EC55, and ST62) or non-resistant (EC39, N9, and ST72)—were applied for the microplate assessment of legumin’s antibacterial properties. All of the species were isolated from our earlier surveys, verified serologically by MALDI-TOF MS (VITEK®MS, database version 3, BioMerieux, France), and assessed quantitatively and phenotypically for antibiotic resistance using the VITEK®-2 system and the Kirby-Bauer disc diffusion method. Data on Salmonella antibiotic susceptibility, including molecular characterization, are available¹²; however, data on Escherichia coli and Staphylococcus aureus are still pending publication. For the challenge experiment, Listeria monocytogenes (ATTC 35152) were obtained from the Animal Health Research Institute (AHRI) in Dokki, Egypt.

Cowpea legumin’s minimum inhibitory concentration (MIC)

Legumin’s MIC was ascertained using the turbidimetric microdilution method based on Resazurin⁶¹. A stock solution containing 50 mg/mL of legumin was generated using sterile distilled water. A 100 µl of this solution was applied to the first column of a 96-well plate, which was subsequently twice serially diluted on Columns 2–10. This resulted in a CPL concentration range of 50–0.1 mg/mL. After that, each well in Columns 1–10 received 50 µl of the previously diluted stock bacterial suspension at 6 log CFU/mL, resulting in a final concentration of 5–5.7 log CFU/mL. Column 12 held 100 µl of the medium broth (as a control to monitor sterility), and Column 11 held 100 µl of the diluted standardized inoculum. Following a 24 h incubation period at 37 °C, resazurin (0.015%) was introduced to each well (30 µl per well), and the wells were then incubated for an additional 2–4 h to determine any color changes. Columns with no color change (blue resazurin color stayed intact) at the end of the incubation period were assessed as being higher than the MIC value⁶¹. By immediately plating the contents of wells with concentrations greater than the MIC value, the minimum biocidal concentration (MBC) was ascertained⁶¹.

Sample preparation and distribution

On the day of slaughter, fresh ribeye loin (Musculus longissimus thoracis et lumborum, LTL) from two beef carcasses were obtained from a local meat retailer and instantly delivered to the laboratory for investigation. LTL steaks weighing 60 ± 5 g (about 3 × 6 cm² and 1.5-cm thick) were aseptically prepared from both sides of each carcass. Then were randomly allocated to one of three treatments: no CPL addition (control), fortification with 1 mg/g of CPL (CPL1), and fortification with 0.5 mg/g of CPL (CPL0.5). The allocation was carefully manipulated to ensure that the two carcasses LTL steaks were included in the three treatments over the six checking chilling days. The individual treatment involved forty-eight steaks divided into two replicates, each consisting of twenty-four pieces. Each replicate was allocated over six checking days, with four steaks per day. LTL-steaks of the CPL1 group were directly supplied with 1 mg/g CPL powder, then 100 mL of sterile distilled water (DW) was added into the zippered package to achieve equal powder dispersion across all steaks, followed by a moderate rotation. After 30 min, the steaks were placed on a sterilized steel sieve to dry. The same procedures were employed for the control group, which just used Sterile DW, but 0.5 mg/g legumin was used in the CPL0.5 treated LTL-steaks. The LTL-steaks were then bagged and refrigerated in the cooling incubator (Binder KB, BINDER GmbH, Tuttlingen, Germany) at 4 ± 0.1 °C. The physicochemical quality of the longissimus was evaluated on days 0, 3, 6, 9, 12, and 15. Water-holding capacity (WHC), purge loss, cooking loss, Warner-Bratzler Shear Force (WBSF), and instrumental color of LTL-steaks were only compared between the Control and CPL1 groups. The shelf-life, pH, and antioxidant stability of LTL-steaks, on the other hand, were evaluated in three separate groups: control (DW, 1^st), CPL1 (1 mg/g CPL, 2^nd), and CPL0.5 (0.5 mg/g CPL, 3^rd). The antibacterial activity and stability of CPL in minced LTL-steaks against artificially inoculated food-borne pathogens were evaluated using four groups: control, CPL1, and CPL0.5, as well as a fourth group of minced LTL-steaks blended with sodium nitrite at 150 PPM as a positive control for synthetic preservatives.

Physicochemical evaluation of longissimus attributes

The Physicochemical properties of LTL-steaks were assessed by applying previously published methods. These characteristics included color parameters (L*, Lightness; a*, Redness; b*, Yellowness; C, Chroma; and h°, Hue angle) as well as pH, WHC, purge loss, cooking loss (CL), Warner-Bratzler shear force (WBSF)⁶².

pH analysis

The LTL samples designated for microbiological and pH assessment were individually tenfold diluted with sterile distilled water and analyzed for pH using pH-meter electrodes (Jenway 3510 pH-meter, Cole-Parmer, Staffordshire, United Kingdom) at each checking point for each holding container. At room temperature, the pH meter was calibrated using three different pH levels (10, 4, and 7) in conjunction with a temperature metal probe. After the microbiological analysis was done, the pH was assessed.

Water-holding capacity estimation

With minor modifications, the low-speed centrifugation techniques outlined by Honikel and Hamm (1994) were used to assess the water-holding capacity (WHC). In brief, a sample of 5 g of intact LTL-steak was centrifuged for 20 min at 10.000 × g and 5 °C in a 15 mL falcon tube filled with glass beads. The LTL-steak was then removed with forceps, dried with filter paper, and weighed again. The WHC was expressed as the percentage of the weight difference between the LTL-steaks before and after the centrifugation.

Purge loss estimation

The purging loss at each checking point is estimated using the percentage of LTL-steak weight loss from the initial weight recorded on the first day of chilling (0, 3, 6, 9, 12, and 15 day)⁶³.

Cooking loss estimation

On each checking day, three samples were subsampled from chilled LTL duplicates to assess cooking loss and shear force. Shear force blocks were randomly assigned to two cook batches based on the replication number. The LTL samples, previously weighed, were individually placed in plastic thermotolerant bags with thin walls and heated for 30 min at 80 °C in a water bath. They were then chilled with tap water to room temperature, cooled in an ice bath to 5 °C, dried, and weighed again. CL is the percentage difference between the raw and cooked weights⁶³.

Warner-Bratzler Shear Force (WBSF) analysis

Using cooked LTL-steak samples, the WBSF was determined using an Instron 3343 Universal Test Device Mono column (Norwood, MA, USA). Perpendicular to the direction of the fiber, the cores of every M. longissimus were severed from the anterior end. The WBSF value, expressed in kilogram force (kgf), was the average measurement of six cores from each steak⁶⁴.

Instrumental color estimation

In the raw LTL-steaks, three colors were assessed using the CR-410 chromometer (Konica Minolta Sensing INC., Osaka, Japan). Chroma Meter set to observer angle of 2°, aperture size of 8.0 mm with closed cone, L*, a*, b* color space, and illuminant D65. Measurements were taken throughout the cut surface of the LTL-steak after it had bloomed for 30 min. Before measurement, the chromameter was calibrated using a reference white tile. These color values were then used to estimate color saturation as (Hue angle (h˚) = arctg b */a *) and color intensity as (C =(a*^² + b*^²) ^^0.5). Increased chroma values signify heightened saturation in the primary hue of the sample. Higher hue angle (or color intensity) values, on the other hand, correspond to less red meat⁶⁵. Six measurements were averaged out for each group.

Microbiological assessment of chilled M. longissimus

Furthermore, the aerobic plate count (APC), coliform count, lactic acid bacteria count, and staphylococcal count of LTL-steak generated from compared groups were assessed over 15 days (1, 3, 6, 9, 12 and 15) in programable cooling incubator (Binder KB, BINDER GmbH, Tuttlingen, Germany) at 4 °C.

Determination of aerobic plate count

The aerobic plate count (APC) in LTL-steaks was assessed the same way as for ground beef products⁶⁶. Each sample was produced as a 10% homogenate, then serially diluted ten times, with 1 mL of each dilution inserted into two separate sterile Petri dishes. After that, the solidified inoculation plates were incubated for 24 h at 37 °C⁶⁷.

Determination of coliform count

One mL of previously prepared tenfold dilutions was inoculated into two independent sterile Petri dishes of Violet red bile agar at 37 °C for coliform enumeration in LTL-steak⁶⁸.

Determination of Staphylococcus count

Staphylococcus counts in LTL-steaks were determined triple using the surface-plating method on the Baird Parker agar plate, as previously published for milk⁶⁹. One milliliter of each of the previously made serial dilutions was distributed using a sterile disposable spreader. The plates were left upright in the incubator for approximately 10 min, or 1 h until the agar absorbed the inoculums. After that, the inoculated were inverted and incubated at 37 °C for 48 h.

Lactic acid bacteria count

Man, Rogosa, and Sharpe medium Agar (MRS, HiMedia, USA) was used to determine LAB, and samples were cultured anaerobically at 30 °C for 72 h⁶⁶.

Cowpea-legumin in-vivo antibacterial activity and stability

CPL was tested in-vivo for antibacterial activity and stability against L. monocytogenes and Salmonella enterica subspecies enterica (N7) in minced LTL. As a positive control for synthetic preservatives, another set of minced LTL-steaks containing sodium nitrite (NaNO₂) at 150 PPM was prepared. The minced LTL-steaks were categorized into four groups: control, CPL1, CPL0.5, and NaNO₂-treated. Minced LTL-steaks of CPL1 and CPL0.5 groups were blended directly with cowpea-legumin at 1 mg/g and 0.5 mg/g, respectively, whereas the fourth group was mixed thoroughly with 150 PPM of NaNO₂. To obtain a final inoculated pathogen initial level of 3 log CFU/g in minced LTL, a fresh working culture of L. monocytogenes and Salmonella enterica (N7) from an overnight culture at 37 °C was serially adjusted to 5 log CFU/mL and injected into the four minced LTL groups at a rate of 2 mL/100 g⁶⁶. Then, 10 g of each group was transferred to a sterile glass bottle (125 mL with rubber closure). Three bottles (three replicates) from each of the four groups were randomly assigned to the six checkpoints (1, 3-, 6-, 9-, 12- and 15-day post-treatment) and then stored at 4 ± 0.1 °C in the incubator (Binder, BINDER GmbH, Tuttlingen, Germany). To undertake in-vivo microbiological investigations, 10 g of the spiked minced LTL-steak was homogenized for one min in a sterile stomacher bag using 90 mL of sterile distilled water (Stomacher 400 R, Seward, UK). Once the homogenization and decimal dilutions were completed, 100 μL was applied to Hektoen enteric agar plates (Condalab, Spain) and Oxford agar (supplied with selective Oxford supplement, Oxoid, Hampshire, United Kingdom) to count Salmonella spp. and L. monocytogenes, respectively. The colonies exhibiting a certain morphology were counted 24 h after the inoculation plates were incubated at 37 ± 0.2 °C.

Malondialdehyde estimation

Using HPLC (Agilent HP 1200 series system, USA) and the guidelines provided by refs. ^70,71, malondialdehyde (MDA) levels in homogenized LTL-steaks were determined. Using an ice-cold homogenizer, a 10% sample homogenate (w/v) and ice-cold 0.1 M Tris-HCl pH 7.4 were created (Glas-Col, USA). The homogenate was centrifuged for 15 min at 2000 × g at 4 °C to remove debris. In addition, 1 mM of stock standard MDA solution was prepared by dissolving 25 μL of 1,1,3,3 tetra-ethoxy-propane (TEP) in 100 mL of water. After that, the 20 nmol/mL working standard was made by dissolving 1 mL of TEP stock solution in 50 mL of 1% sulfuric acid for 2 h at room temperature, which was then diluted with 1% sulfuric acid to yield a final standard concentration of 1.25 nmol/mL for total MDA analysis. The results were expressed as nM/g.

Statistical analyses

The data analysis was conducted using SPSS Version 22. (SPSS Inc. Chicago, IL, USA). The impact of treatments (control, CPL1), chilling periods (1-, 3-, 6-, 9-, 12-, and 15-day), and their interaction on LTL-steaks quality metrics was studied using general linear mixed models (GLM). LTL-steaks were considered random, whereas treatments and chilling storage time were fixed variables. Similar statistical techniques were used for WBSF and cooking loss; however, the random model included cooking batches. To assess LTL-steaks microbiological, antioxidant, and pH characteristics, models included fixed effects for treatments (control, CPL1, and CPL0.5) and chilling storage durations (1, 3, 6, 9, 12, and 15 days), random terms for LTL-steaks, and interaction effects (treatment × chilling day). In-vivo CPL antibacterial activity was examined using fixed effects of treatment (control, CPL1, CPL0.5, and nitrite) and chilling storage durations (1, 3, 6, 9, 12, and 15 days), random terms for minced LTL-steaks bottles, and an interaction term between treatment and chilling day. The results are shown with their means and the overall standard errors of the means. The statistical model employed Tukey’s b multiple comparison test to quantify the effects of CPL and their levels, as well as the impacts of NaNO₂ compared to control, and to assess unique monitoring point averages within the same group. Significant differences were determined with a p-value of less than 0.05.

Author contributions

Wessam Dawam: Conceptualization, Investigation, Data curation, Writing- Original draft preparation and Visualization. Shimaa Edris: Conceptualization, Supervision, Investigation, Writing- Original draft, Validation, Visualization, Reviewing and Editing. Mai Elsheikh: Conceptualization, Investigation, Data curation, Writing- Original draft preparation and Visualization. Ali Osman: Conceptualization, Supervision, Original draft, Validation, Visualization, Reviewing and Editing. Ahmed Hamad: Visualization, Reviewing and Editing. Mahmoud Sitohy: Conceptualization, Supervision, Original draft, Validation, Visualization, Reviewing and Editing. Islam Sabeq: Conceptualization, Supervision, Investigation, Software, Validation, Visualization, Writing, Reviewing and Editing.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

All materials described in the manuscript, including all relevant raw data, were included as main and supplementary tables and figures. The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

[CR1] 1.OECD-FAO. World meat projections. OECD-FAO Agricultural Outlook 2021-2030, 10.1787/cbc9005c-en (2021).

[CR2] 2.Zhou, J., Li, D., Zhang, X., Liu, C. & Chen, Y. Valorization of protein-rich waste and its application. Sci. Total Environ.901, 166141 (2023). [DOI] [PubMed] [Google Scholar]

[CR3] 3.Zhu, X. et al. The Impacts of Different Pea Protein Isolate Levels on Functional, Instrumental and Textural Quality Parameters of Duck Meat Batters. Foods11, 1620 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Falowo, A. B., Fayemi, P. O. & Muchenje, V. Natural antioxidants against lipid–protein oxidative deterioration in meat and meat products: A review. Food Res. Int.64, 171–181 (2014). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Manessis, G. et al. Plant-Derived Natural Antioxidants in Meat and Meat Products. Antioxidants9, 1215 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Jeremić, S. et al. Importance of hydrogen bonding and aromaticity indices in QSAR modeling of the antioxidative capacity of selected (poly)phenolic antioxidants. J. Mol. Graph. Model.72, 240–245 (2017). [DOI] [PubMed] [Google Scholar]

[CR7] 7.Wang, W. et al. Analysis, occurrence, toxicity and environmental health risks of synthetic phenolic antioxidants: A review. Environ. Res.201, 111531 (2021). [DOI] [PubMed] [Google Scholar]

[CR8] 8.Zhang, D. et al. Recent trends in the micro-encapsulation of plant-derived compounds and their specific application in meat as antioxidants and antimicrobials. Meat Sci.191, 108842 (2022). [DOI] [PubMed] [Google Scholar]

[CR9] 9.National Academies of Sciences, Engineering, and Medicine. Combating Antimicrobial Resistance and Protecting the Miracle of Modern Medicine (National Academies Press (US), 2021). [PubMed]

[CR10] 10.EFSA Panel on Biological Hazards (BIOHAZ). et al. Transmission of antimicrobial resistance (AMR) during animal transport. EFSA J. 20, e07586–e07586 (2022). [DOI] [PMC free article] [PubMed]

[CR11] 11.Samreen, Ahmad, I., Malak, H. A. & Abulreesh, H. H. Environmental antimicrobial resistance and its drivers: a potential threat to public health. J. Glob. Antimicrobial Resistance27, 101–111 (2021). [DOI] [PubMed] [Google Scholar]

[CR12] 12.Sabeq, I. et al. Prevalence and molecular characterization of foodborne and human‐derived Salmonella strains for resistance to critically important antibiotics. Transbound. Emerg. Dis.69, e2153–e2163 (2022). [DOI] [PubMed] [Google Scholar]

[CR13] 13.World Health Organization. A one health priority research agenda for antimicrobial resistance (WHO, 2023).

[CR14] 14.Abdel-Shafi, S. et al. Biochemical, biological characteristics and antibacterial activity of glycoprotein extracted from the epidermal mucus of African catfish (Clarias gariepinus). Int. J. Biol. Macromolecules138, 773–780 (2019). [DOI] [PubMed] [Google Scholar]

[CR15] 15.El‐Zahar, K., Chobert, J., Sitohy, M., Dalgalarrondo, M. & Haertlé, T. Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage. Food/. Nahrung47, 199–206 (2003). [DOI] [PubMed] [Google Scholar]

[CR16] 16.Pina-Pérez, M. C. & Ferrús Pérez, M. A. Antimicrobial potential of legume extracts against foodborne pathogens: A review. Trends Food Sci. Technol.72, 114–124 (2018). [Google Scholar]

[CR17] 17.Osman, A., Daidamony, G., Sitohy, M., Khalifa, M. & Enan, G. Soybean glycinin basic subunit inhibits methicillin resistant-vancomycin intermediate Staphylococcus aureus (MRSA-VISA) in vitro. Int. J. Appl. Res. Nat. Products9, 17–26 (2016). [Google Scholar]

[CR18] 18.Pathiraje, D., Carlin, J., Der, T., Wanasundara, J. P. D. & Shand, P. J. Generating Multi-Functional Pulse Ingredients for Processed Meat Products-Scientific Evaluation of Infrared-Treated Lentils. Foods12, 1722 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Gondwe, T. M., Alamu, E. O., Mdziniso, P. & Maziya-Dixon, B. Cowpea (Vigna unguiculata (L.) Walp) for food security: an evaluation of end-user traits of improved varieties in Swaziland. Sci. Rep.9, 15991–15991 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Osman, A., Goda, H. A. & Sitohy, M. Storage stability of minced beef supplemented with chickpea legumin at 4°C as a potential substitute for nisin. LWT93, 434–441 (2018). [Google Scholar]

[CR21] 21.Abdel-Shafi, S. et al. Characterization and Antibacterial Activity of 7S and 11S Globulins Isolated from Cowpea Seed Protein. Molecules24, 1082–1097 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

[CR22] 22.Ebrahim, A. E. et al. Antibacterial activity of bioactive compounds extracted from red kidney bean (Phaseolus vulgaris L.) seeds against multidrug-resistant Enterobacterales. Front Microbiol13, 1–17 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Sitohy, M., Mahgoub, S. & Osman, A. Controlling psychrotrophic bacteria in raw buffalo milk preserved at 4 °C with esterified legume proteins. LWT - Food Sci. Technol.44, 1697–1702 (2011). [Google Scholar]

[CR24] 24.Murray, C. J. et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet399, 629–655 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Bertrand, R. L. Lag Phase Is a Dynamic, Organized, Adaptive, and Evolvable Period That Prepares Bacteria for Cell Division. J. Bacteriol.201, e00697–18 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Jeleníková, J., Pipek, P. & Staruch, L. The influence of ante-mortem treatment on relationship between pH and tenderness of beef. Meat Sci.80, 870–874 (2008). [DOI] [PubMed] [Google Scholar]

[CR27] 27.Warner, R. D. The Eating Quality of Meat—IV Water-Holding Capacity and Juiciness. Lawries Meat Sci. 419–459, 10.1016/b978-0-08-100694-8.00014-5 (2017).

[CR28] 28.Morrissey, P. A. & Kerry, J. P. Lipid oxidation and the shelf-life of muscle foods. In Understanding and Measuring the Shelf-Life of Food 357–395, 10.1533/9781855739024.2.357 (2004).

[CR29] 29.O’Kane, F. E., Vereijken, J. M., Gruppen, H. & Van Boekel, M. A. J. S. Gelation Behavior of Protein Isolates Extracted from 5 Cultivars ofPisum sativumL. J. Food Sci.70, C132–C137 (2005). [Google Scholar]

[CR30] 30.Youssef, M. K. & Barbut, S. Effects of two types of soy protein isolates, native and preheated whey protein isolates on emulsified meat batters prepared at different protein levels. Meat Sci.87, 54–60 (2011). [DOI] [PubMed] [Google Scholar]

[CR31] 31.Zhang, H., Takenaka, M. & Isobe, S. DSC and electrophoretic studies on soymilk protein denaturation. J. Therm. Anal. Calorim.75, 719–726 (2004). [Google Scholar]

[CR32] 32.Kilara, A. Interactions of Ingredients in Food Systems. Food Sci. Technol. 1–20, 10.1201/9781420028133.ch1 (2005).

[CR33] 33.Dunshea, F. et al. Meat Tenderness: Underlying Mechanisms, Instrumental Measurement, and Sensory Assessment. Meat Muscle Biol.4, 17 (2021).

[CR34] 34.Bolumar, T. & Toepfl, S. Application of Shockwaves for Meat Tenderization. Innovative Food Processing Technol. 231–258, 10.1016/b978-0-08-100294-0.00009-2 (2016).

[CR35] 35.Zhang, W. & Liu, R. Molecular factors influencing meat tenderness. Encyclopedia Meat Sci. 306–312, 10.1016/b978-0-323-85125-1.00153-8 (2024).

[CR36] 36.Lomiwes, D., Farouk, M. M., Frost, D. A., Dobbie, P. M. & Young, O. A. Small heat shock proteins and toughness in intermediate pHu beef. Meat Sci.95, 472–479 (2013). [DOI] [PubMed] [Google Scholar]

[CR37] 37.Silva, J. A., Patarata, L. & Martins, C. Influence of ultimate pH on bovine meat tenderness during ageing. Meat Sci.52, 453–459 (1999). [DOI] [PubMed] [Google Scholar]

[CR38] 38.Hamoen, J. R., Vollebregt, H. M. & van der Sman, R. G. M. Prediction of the time evolution of pH in meat. Food Chem.141, 2363–2372 (2013). [DOI] [PubMed] [Google Scholar]

[CR39] 39.Destefanis, G., Brugiapaglia, A., Barge, M. T. & Dal Molin, E. Relationship between beef consumer tenderness perception and Warner–Bratzler shear force. Meat Sci.78, 153–156 (2008). [DOI] [PubMed] [Google Scholar]

[CR40] 40.Akesowan, A. Quality characteristics of light pork burgers fortified with soy protein isolate. Food Sci. Biotechnol.19, 1143–1149 (2010). [Google Scholar]

[CR41] 41.Elsheikh, M. et al. Soybean Glycinin’s Antibacterial Properties Provide a Feasible Natural Alternative for Improving the Overall Quality and Shelf-Life of Beef Steaks and Combating Foodborne Pathogens. Food Bioprocess Technol.10.1007/s11947-024-03557-z (2024).

[CR42] 42.Elsheikh, M. et al. Cowpea 11S globulin spray quadruples shrimp shelf-life and maintains freshness. Benha Vet. Med. J.46, 74–79 (2024). [Google Scholar]

[CR43] 43.Sánchez-Escalante, A., Djenane, D., Torrescano, G., Beltrán, J. A. & Roncalés, P. The effects of ascorbic acid, taurine, carnosine and rosemary powder on colour and lipid stability of beef patties packaged in modified atmosphere. Meat Sci.58, 421–429 (2001). [DOI] [PubMed] [Google Scholar]

[CR44] 44.Ju, M. et al. A novel pickering emulsion produced using soy protein-anthocyanin complex nanoparticles. Food Hydrocoll.99, 105329 (2020). [Google Scholar]

[CR45] 45.Zheng, J., Zheng, X., Zhao, L., Yi, J. & Cai, S. Effects and interaction mechanism of soybean 7S and 11S globulins on anthocyanin stability and antioxidant activity during in vitro simulated digestion. Curr. Res. Food Sci.4, 543–550 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Kalita, C. et al. Recent trends in antioxidative peptides derived from soybean and other soy-based products: A comprehensive review. Process Biochem.136, 311–323 (2024). [Google Scholar]

[CR47] 47.Zhao, J., Xiong, Y. L. & McNear, D. H. Changes in structural characteristics of antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals. J. Food Sci.78, C152–C159 (2013). [DOI] [PubMed] [Google Scholar]

[CR48] 48.Anton, D. et al. Antimicrobial and Antioxidative Effects of Plant Powders in Raw and Cooked Minced Pork. Foods8, 661 (2019). [DOI] [PMC free article] [PubMed]

[CR49] 49.Björkroth, J. Microbiological ecology of marinated meat products. Meat Sci.70, 477–480 (2005). [DOI] [PubMed] [Google Scholar]

[CR50] 50.Zhou, G., Xu, X. & Liu, Y. Preservation technologies for fresh meat–A review. Meat Sci.86, 119–128 (2010). [DOI] [PubMed] [Google Scholar]

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Cowpea legumin preservative impacts on beef ribeye and implications on antibiotic resistant food borne pathogens

Wesam Dawam

Shimaa Edris

Ali Osman

Mai Elsheikh

Ahmed Hamad

Mahmoud Sitohy

Islam Sabeq

Abstract

Introduction

Results

Table 1.

Table 2.

Table 3.

Discussion

Methods

Experiment management and approval

Cowpea 11S globulin (legumin) protein extraction

The preparation of pathogens and inoculums

Cowpea legumin’s minimum inhibitory concentration (MIC)

Sample preparation and distribution

Physicochemical evaluation of longissimus attributes

pH analysis

Water-holding capacity estimation

Purge loss estimation

Cooking loss estimation

Warner-Bratzler Shear Force (WBSF) analysis

Instrumental color estimation

Microbiological assessment of chilled M. longissimus

Determination of aerobic plate count

Determination of coliform count

Determination of Staphylococcus count

Lactic acid bacteria count

Cowpea-legumin in-vivo antibacterial activity and stability

Malondialdehyde estimation

Statistical analyses

Author contributions

Funding

Data availability

Competing interests

Footnotes

References

Associated Data

Data Availability Statement

ACTIONS

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RESOURCES

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