The Effect of Combined Extracts from By-Products, Seaweed, and Pure Phenolics on the Quality of Vacuum-Packed Fish Burgers

Abstract

The objective of the present study was to determine the effect of mixed plant extracts on chemical (pH, total volatile base nitrogen (TVB-N), trimethylamine nitrogen (TMA), thiobarbituric acid reactive substances (TBARS), biogenic amines, relative fatty acid composition) and microbiological quality indicators of vacuum-packed fish burgers stored at 0 ± 2 °C over 13 days. Three mixtures of common juniper by-product and blackberry leaves extracts (JB), Padina pavonica and prickly juniper needles extracts (PCJ), and blackberry leaves extract with catechin and vanillic (BCV) were tested. At the end of storage, TVB-N (15.38–20.03 mg/100 g) and TMA (10.64–15.63 mg/100 g) of burgers with extracts were significantly lower than those of the control group (22.77 mg TVB-N/100 g, 18.37 mg TMA/100 g). The TBARS values in the control burger reached 2.62 ± 0.02 µmol malondialdehyde (MDA)/100 g, while in burgers with extracts, final values were in the range of 0.62 ± 0.01 to 0.80 ± 0.02 µmol MDA/100 g. The extracts showed no effect on biogenic amine formation (tryptamine, putrescine, and cadaverine levels increased during the storage, being the lowest in BCV) or microbial counts, with the exception of the Pseudomonas sp. counts that were significantly lower in JB and PCJ in comparison to the control, reaching 4.1, 4.1, and 5.0 log CFU/g in JB, PCJ, and control, respectively.

1. Introduction

Fish are an excellent source of high-quality proteins, essential minerals such as iron, iodine, zinc, selenium, and potassium, as well as vitamins including D and B2 (riboflavin). They are also the primary dietary source of long-chain polyunsaturated omega-3 fatty acids (PUFAs), while being naturally low in cholesterol and saturated fats. Due to this unique nutritional profile, fish are widely recognized as an important component of a healthy diet. Regular consumption has been associated with a range of health benefits, including the prevention of chronic diseases, reduced inflammation, enhanced wound healing, and improved cardiovascular and neurological health [1,2].

Despite the described health benefits, fish consumption remains below recommended levels in many countries, including Croatia, where dietary guidelines advise at least two servings per week. Barriers to regular consumption include the high cost and limited availability of fresh fish, as well as the general perception of fish as an inconvenient food, which requires more time for purchasing, preparation, and consumption [3]. Therefore, there is a need for the seafood industry to innovate and adapt to the requirements of consumers by offering products that are more convenient, appealing, and ready-to-cook, with clear labeling and appropriate portion sizes to meet modern consumer expectations.

A major challenge for seafood producers is the fact that fresh fish is a highly perishable commodity with a very short shelf-life of only 10–18 days in the cold distribution chain [4]. The principal causes of quality and safety deterioration of fish are enzymatic activity, microbial proliferation, and chemical spoilage (mainly oxidation) [5]. In recent years, the use of natural additives—derived from plants, algae, and bacteria—has gained attention as a promising strategy to inhibit these spoilage mechanisms. These natural compounds can delay microbial growth, enzymatic degradation, and lipid oxidation, thereby extending shelf-life while also offering potential health benefits [6]. With increasing consumer preference for natural over synthetic additives, the development of functional seafood products enriched with such bioactive compounds is gaining momentum [7,8].

One promising approach involves the incorporation of natural extracts into fresh fish products, such as fish burgers. Numerous studies have investigated the effects of plant-derived extracts and essential oils—such as those from sage, rosemary, thyme, oregano, cinnamon, lemon balm, and clove—on the preservation, sensory characteristics, and nutritional quality of fish products [9,10,11]. These extracts are rich in phenolic compounds known for their antioxidant and antimicrobial activities. Their effectiveness has been demonstrated in both fresh and smoked fish fillets [12,13,14,15,16,17], and to a lesser extent, in fish burgers [18,19,20,21]. Furthermore, combining these extracts with preservation techniques such as modified atmosphere packaging (MAP) or edible coatings has proven effective in prolonging the shelf-life of fish stored at 2–4 °C [9,10,11].

Beyond well-known herbs, recent research has expanded to include lesser-known plant sources such as Juniperus species from the Mediterranean maquis [22], marine macro- and microalgae [23,24,25,26], and agro-food by-products [27]. Despite this progress, most studies have focused on fish fillets, with limited research addressing their application in processed products like fish burgers. Some notable exceptions include the use of broccoli and pomegranate by-products in mackerel and hake burgers [28,29], and olive leaf extract for extending the shelf-life of vacuum-packed salmon burgers [30].

In our previous research, we demonstrated the antioxidant and antimicrobial potential of selected Mediterranean plants, seaweeds, and agro-food by-products. We also investigated potential synergistic effects when combining these extracts with pure phenolic compounds [22,27,28]. Building on these findings, the current study aimed to evaluate the most promising extracts in a food model. Specifically, we developed fresh fish burgers supplemented with: (i) a mixture of common juniper (Juniperus communis) by-products and blackberry (Rubus fruticosus) leaves; (ii) a combination of brown seaweed (Padina pavonica) and prickly juniper (J. oxycedrus) needles; and (iii) blackberry leaf extract enriched with catechin and vanillic acid. These formulations were assessed for their effects on the chemical and microbial quality of vacuum-packed fish burgers.

2. Materials and Methods

2.1. Preparation of Natural Extracts and Pure Compounds

Blackberry (Rubus fruticosus) leaves, prickly juniper (Juniperus oxycedrus) needle and common juniper (J. communis) by-products, and seaweed Padina pavonica extracts were prepared following the procedure in Barbieri et al. [22]. Briefly, plant material was shade-dried and extracted using microwave-assisted extraction (5 min, 600 W, 50% ethanol), while Padina pavonica raw material was freeze-dried and extracted with 50% ethanol using microwave-assisted extraction (15 min, 200 W). After the extraction, ethanol was evaporated, and the remaining water was freeze-dried. Based on the preliminary results [22,25,26,27], the dried extracts and pure compounds (catechin and vanillic acid bought from Sigma Aldrich, St. Louis, MO, USA) were mixed and dissolved in distilled water before their addition to minced meat (

Table 1. Composition of fish burger samples: Control and formulations containing natural extracts and pure compounds.

Fish Burger Sample	Abbreviation
Burger without natural extracts (control)	CON
Burger with 2% (w/w) common juniper by-product and blackberry leaf extract (1:2 ratio)	JB
Burger with 2% (w/w) Padina pavonica and prickly juniper needle extract (1:1 ratio)	PCJ
Burger with 2% (w/w) blackberry leaf extract, catechin, and vanillic acid (2:1:1 ratio)	BCV

). The concentrations of natural extracts added to burgers were 2% (w/w), chosen based on the preliminary results, taking into account in vitro activity. Each extract was first evaluated individually, followed by testing in various combinations and concentrations. The most effective ratios were identified based on observed synergistic, additive, or antagonistic interactions, as well as their effect on the sensory properties.

2.2. Preparation of Fish Burgers

In collaboration with a local fish processor (Centaurus Ltd., Solin, Croatia), fish burgers were produced following the processing steps shown in Figure 1. Upon arrival at the factory, fresh hake (Merluccius merluccius) and sea bass (Dicentrarchus labrax) were filleted, and the fillets were minced in a 1:1 ratio, with the addition of 1% salt (w/w). The proximate composition of the resulting minced fish–salt mixture (used as the control sample) was determined using the following methods: The total lipid content was measured according to the method of Bligh and Dyer [29]; crude protein (Kjeldhal method, N × 6.25), water content (drying the samples at 105 °C to constant weight), crude ash (calcination at temperatures ≤ 500 °C), and NaCl content (volumetric method with silver nitrate standard solution) were analyzed following AOAC protocols [30].

The minced fish base was divided into four groups, as shown in Table 1. The original mixture (minced fish meat and salt) served as the control, while the remaining three groups were supplemented with natural extracts, which were manually mixed into the base. The burgers were formed using a manual burger press (model PHR130, SAP, Bologna Italy), vacuum-packed, and stored at 0 ± 2 °C for 13 days. Sampling was conducted on days 0, 1, 3, 5, 7, 9, 11, and 13. During the storage period, samples were analyzed for pH, thiobarbituric acid reactive substances (TBARS), total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA), biogenic amines, microbiological load (starting from day 1), and relative fatty acid composition (analyzed at the beginning and end of the storage).

2.3. Chemical Analyses

For the pH determination, 10 g of fish burger was weighed and homogenized with 10 mL of distilled water. A digital pH meter (Hanna Instruments, Woonsocket, RI, USA) was used for the measurement [31].

The thiobarbituric reactive substances assay (TBARS) was used for the determination of lipid oxidation in fish burgers [32,33]. Briefly, 20 g of the fish burger sample was homogenized with 40 mL of 7.5% (v/v) trichloroacetic acid. Then, 5 mL of homogenate was filtered and combined with 5 mL of 2-thiobarbituric acid (0.02 M) and incubated for 40 min at 95 °C. The absorbance of the mixture was measured at 538 nm. A standard curve was plotted with 1,1,3,3-tetraethoxypropane, and results were expressed as µmol malondialdehyde (MDA)/100 g.

TVB-N and TMA were analyzed using a Kjeldahl distillation unit (model B-324, Büchi AG, Flawil, Switzerland) by the method described in Šimat et al. [34]. Briefly, 100 g of fish burgers were homogenized with 7.5% (v/v) trichloroacetic acid and centrifuged for 10 min at 4500× g. The supernatants were filtered and distilled. The distillates were titrated using automatic titration (702 SET/MET titrino, Metrohm AG, Bangkok, Thailand). The results were expressed in mg/100 g.

A profile of eight biogenic amines (β-phenylethylamine, cadaverine, histamine, putrescine, spermidine, spermine, tryptamine, and tyramine) was determined using a high-performance liquid chromatography (HPLC, Agilent 1200 Series LC system, Agilent Technologies Inc., Waldbronn, Germany) method of Eerola et al. [35], modified by Šimat and Dalgaard [36]. The standards were purchased from SigmaAldrich (St. Louis, MO, USA), the analyses were done in duplicate, and the results were expressed in mg/kg of the sample.

2.4. Microbiological Analyses

For microbiological analyses, 25 g of fish burger samples were homogenized (Masticator, IUL S.A., Barcelona, Spain) with 250 mL of buffered peptone water (BPW, Biolife Italiana, Milano, Italy), and serial dilutions were prepared. Samples were inoculated in triplicate on Plate Count Agar (PCA, Biolife Italiana) for total counts of aerobic mesophilic bacteria (MEZ) and total counts of aerobic psychrophilic bacteria mesophilic (PSI), on Violet Red Bile Glucose Agar (VRBG, Biolife Italiana) for enumeration of Enterobacteriaceae, on MRS Agar with Tween 80 (MRS, Biolife Italiana) for lactic acid bacteria (LAB), and on Pseudomonas Agar Base (PAB, Biolife Italiana) for enumeration of Pseudomonas sp. PCA plates were incubated at 7 °C for 10 days and at 30 °C for 72 h, VRBG and PAB at 30 °C for 48 h, and MRS plates were vacuum packed and incubated at 30 °C for 72 h. The number of colony-forming units (CFU) per gram of sample was expressed as log CFU/g. The analyses were conducted in triplicate.

2.5. Lipid Extraction and Determination of Fatty Acid Profile

The lipid content in the fish burgers was determined on days 1 and 11 of storage according to a slightly modified method described by the Association of Official Analytical Chemists (AOAC) Official Method 948.15 [37]. A mass of 5 g of homogenized samples was mixed with 10 mL of 8 M HCl in a 50 mL centrifuge tube. The samples were digested at 100 °C for 90 min in a water bath (with vortexing after 45 min of digestion). After digestion, 5 mL of methanol was added to the cooled samples and vortexed for 30 s. For the extraction of the lipid content, 15 mL of diethyl ether was added, and the samples were shaken vigorously for 1 min. Then, a 15 mL aliquot of petroleum ether was added, and the samples were shaken for 20 s. The mixed samples were centrifuged at 1200 rpm for 5 min, and the upper ether-fat layer was transferred to a weighed flask, which was stored in a desiccator. The extraction step was repeated three times. The lipid content was obtained by evaporation on a rotary evaporator at 60 °C, and the flask was cooled in the desiccator at room temperature in the dark. The collected lipid content (0.1 g) was dissolved in heptane (2 mL) and stored in glass vials at 4 °C.

The fatty acid profile of the fish burgers was determined according to the method described by Šimat et al. [38]. The fatty acid methyl esters (FAMEs) were prepared by adding 2M KOH in methanol (0.2 mL) and analyzed by GC-FID using a capillary column RTX 2560 (100 m × 0.25 mm i.d.; coating thickness 0.25 mm; Restek, Bellefonte, PA, USA). A volume of 1 µL of the heptane layer was injected into the chromatograph at a split ratio of 1:100. The flow rate of the carrier gas helium was 3 mL/min, while the temperature of the injector and detector was 225 °C and 240 °C, respectively. The initial temperature of 140 °C was maintained for 5 min, then increased to 240 °C at a rate of 4 °C/min and maintained at 240 °C for 20 min. The FAMEs were identified by comparison with standards (Supelco 37 Component FAME Mix, Sigma–Aldrich, St. Louis, MO, USA). Results are expressed as the percentage of methyl esters of specific fatty acids and determined by the ratio of the peak area of interest to the total area of all peaks. All samples were analyzed in duplicate.

2.6. Statistical Analysis

The graphs were constructed using Microsoft Excel (Microsoft Office Professional Plus 2019, Version 1808). The statistical differences between the control burger and burgers with extracts were evaluated using analyses of variance (one-way ANOVA followed by Fisher’s least significant difference test). The analyses were performed using Statgraphics Centurion-Ver. 16.1.11 (StatPoint Technologies, Inc., Warrenton, VA, USA) with a significance level set at 0.05.

3. Results and Discussion

The proximate composition of the base used for burger production was as follows: water content 77.69 ± 0.06 g/100 g, protein content 17.00 ± 0.04 g/100 g, lipid content 2.92 ± 0.05 g/100 g, ash 1.05 ± 0.07 g/100 g, and 0.96 ± 0.01 g/100 g of NaCl.

To evaluate the effect of natural extracts and pure compounds on vacuum-packed fish burgers, the pH, TBARS, TVB-N, TMA, biogenic amine profile, microbiological analyses (total mesophilic and psychrophilic microbial counts, Enterobacteriaceae, Pseudomonas sp., and lactic acid bacteria), and fatty acid composition were analyzed over 13 days of storage. The burger was chosen as the food model for this study to achieve better and more even distribution of the extracts in the product.

3.1. The Results of Chemical Analyses

The change in pH in fish burgers during the shelf-life period is shown in Figure 2. The pH of the control burger ranged from 7.05 ± 0.04 to 7.16 ± 0.00. Burgers with extracts maintained lower pH values compared to the control. The pH values recorded for JB and PCJ burgers increased from 6.87 ± 0.01 to 6.89 ± 0.01 and from 6.91 ± 0.02 to 6.93 ± 0.02 during the storage period, respectively. The lowest pH was recorded for the BCV burger and ranged from 6.51 ± 0.01 to 6.62 ± 0.00, suggesting that the extract may have acidifying properties. These results are in agreement with the pH values reported for sea bass fillets treated with plant extracts and packed under a modified atmosphere. The authors reported a pH between 6.45 and 7.11 during the storage period of 20 days [16,39].

Spoilage through lipid oxidation is one of the most common forms of fish product deterioration. The burgers were made from fresh, lean fish with lipid content of <3%, and they were vacuum-packed and stored at a low temperature. Both hake and sea bass have low initial TBARS values, usually in the range of 0.3–0.9 µmol MDA per 100 g of fish [40]. The TBARS results for fish burgers are shown in Figure 3. In comparison to the control, all additions inhibited the lipid oxidation of the burgers and their TBARS values stayed below the acceptable limit for MDA content of 1.4–2.8 µmol MDA/100 g of fish throughout the storage period [40]. A significant increase in TBARS was observed only in the control samples, from 0.58 ± 0.00 µmol MDA/100 g at the beginning of the storage period to 2.62 ± 0.02 µmol MDA/100 g at the end of the shelf-life period (Figure 3). The final recorded TBARS values were 0.80 ± 0.02 µmol MA/100 g for JB burgers, 0.62 ± 0.01 µmol MA/100 g for PCJ burgers, and 0.71 ± 0.01 µmol MA/100 g for BCV burgers. Lipid oxidation in fish is a complex process, but it can be concluded that the presence of natural antioxidants (plant, algae extracts, and pure phenolics) showed a protective effect in terms of lipid oxidation [9,41,42]. Brown algae like Padina are rich in polyphenols (e.g., phlorotannins) and have shown excellent antioxidant activity [28] as have other studied extracts [22,27,28,43,44]. The content of total phenolic compounds and activity of the dominant phenolics have previously been correlated with the antioxidant power of plants and algae [25,27,28,45]. The original scientific studies of their direct application in fish burgers or similar products are very limited. Ozogul et al. [16] found that Padina (and other extracts) in MAP sea bass prevented lipid oxidation over 18 days of storage and had the ability to exhibit ~64% radical scavenging activity when incorporated into coatings/films [28]. Juniper species also contain antioxidants (e.g., flavonoids, essential oils) that, in the form of essential oil, demonstrated an antioxidant effect in trout fillets. In the control samples, initial TBARS value rose from ~1.34 mg MDA/kg to 5.67 mg/kg in air-packaged products, but only to 3.74 mg/kg in vacuum + juniper (0.6%) samples [46]. Catechin-rich extracts were previously shown to be effective antioxidants in fish. Tea catechins prevent lipid oxidation in fish mince, lowering TBARS during chilled and frozen storage [47]. Uçak [48] observed that adding 0.5–1% of pomegranate seed extract (rich in catechin-like tannins) to fish burgers significantly lowered TBARS during 8-day chilled storage in comparison to control burgers.

TVB-N and TMA results are shown in Figure 4 and Figure 5. They were used as indicators of quality degradation and spoilage of fish burgers due to bacterial activity and amino acid decarboxylation. The initial TVB-N of the control burger was 14.71 ± 0.10 mg/100 g, while burgers with extracts had significantly lower initial TVB-N, ranging from 13.64 ± 0.02 to 14.35 ± 0.14 mg/100 g. A slight increase was recorded in all burgers at the end of the storage period. However, the control burger had the highest values, 22.77 ± 0.19 mg/100 g, while JB and BCV burgers had lower final values than the initial values for the control burger, at 16.89 ± 0.21 mg/100 g and 15.38 ± 0.45 mg/100 g, respectively. A similar trend was also observed for the TMA values. The control burger’s initial TMA (8.37 ± 0.15 mg/100 g) was insignificantly higher than the initial TMA for burgers with extracts. These values are also higher than those once reported for high-quality fresh fish of <2 mg/100 g [49]. TVB-N and TMA were previously reported to be <60 mgTVB-N/100 g and <40 mg TMA/kg in hake with high initial microbial loads and during storage at 4 °C [50]. On the other hand, the shelf life of whole ungutted hake in ice was established at 8 to 10 days, and TMA values of 5 mg/100 g were proposed for an unacceptable grade of quality [49]. The difference from our results could be because of the mix of two different species and the addition of salt to the minced meat. At the end of the storage period, the control burger had the highest TMA value, 18.37 ± 0.38 mg/100 g, while the lowest TMA was recorded for the BCV burger. The BCV burger had a lower final TMA value (10.64 ± 0.28 mg/100 g) than the initial TMA value for the control burger.

Typically, TVB-N and TMA values vary depending on the size of the fish, its microbiological quality, as well as handling and storage conditions [16,40]. The results showed that these natural extracts slow the accumulation of TVB-N and TMA in fish, preserving the fish muscle’s nitrogenous compounds from degradation. There are a limited number of studies that investigated these particular extracts. In MAP-packed sea bass fillets treated with the Padina extract, TVB-N and TMA values were significantly lower compared to untreated controls [16]. Özpolat [46] reported that juniper oil and vacuum packaging kept TVB-N and TMA below spoilage limits far longer than in untreated fish. Initial TVB-N in trout fillets was 12 mg/100 g, and juniper-treated samples stayed under the acceptable spoilage limit of 25–30 mg/100 g for the entire 28-day storage, whereas controls exceeded this limit much earlier and treated fillets did not reach the typical spoilage odor, correlated with TMA. Additionally, tea polyphenols significantly delayed the formation of volatile bases in carp fillets [47].

Biogenic amines are organic compounds formed through the microbial decarboxylation of amino acids in various foods. At high levels, biogenic amines in fish products can pose serious health risks to consumers. In addition, they are an indicator of food spoilage, improper handling, poor hygiene, and microbial contamination [51]. The results for biogenic amine production in fish burgers with natural extracts and pure compounds during the 13 days of storage are shown in

Table 2. The biogenic amine production (in mg/kg) in fish burgers during 13 days of storage.

Treatment Group	Storage Time	Biogenic Amine (mg/kg)
		β-Phenethylamine	Tryptamine	Putrescine	Cadaverine	Histamine	Tyramine	Spermidine	Spermine
CON	0	6.44 ± 0.19 a	0.24 ± 0.05 a	0.52 ± 0.10 a	0.78 ± 0.07 a	0.00 ± 0.00	0.00 ± 0.00	0.84 ± 0.02 a	2.12 ± 0.08 a
	1	4.58 ± 0.13 b	0.18 ± 0.02 a	0.46 ± 0.08 a	0.89 ± 0.09 a	0.00 ± 0.00	0.62 ± 0.10 a	0.82 ± 0.03 a	1.48 ± 0.11 b
	3	4.82 ± 0.11 b	1.22 ± 0.11 b	0.48 ± 0.05 a	1.22 ± 0.12 b	0.00 ± 0.00	1.80 ± 0.13 b	0.64 ± 0.02 a	2.12 ± 0.10 a
	5	5.24 ± 0.22 a	3.54 ± 0.09 c	0.61 ± 0.07 a	2.49 ± 0.13 c	0.00 ± 0.00	0.90 ± 0.10 a	0.87 ± 0.02 a	2.08 ± 0.13 a
	7	1.10 ± 0.27 c	3.71 ± 0.54 c	0.92 ± 0.04 b	2.52 ± 0.08 c	0.00 ± 0.00	0.78 ± 0.11 a	0.01 ± 0.01 b	1.19 ± 0.06 b
	9	1.38 ± 0.32 c	3.14 ± 0.48 c	0.98 ± 0.15 b	2.50 ± 0.19 c	0.00 ± 0.00	0.58 ± 0,09 a	0.00 ± 0.00	1.39 ± 0.05 a
	11	0.29 ± 0.06 d	3.27 ± 0.38 c	1.44 ± 0.11 c	3.66 ± 0.03 d	0.00 ± 0.00	0,67 ± 0.08 a	0.00 ± 0.00	1.28 ± 0.03 a
	13	0.81 ± 0.18 d	6.34 ± 0.41 d	3.05 ± 0.06 d	3.61 ± 0.00 d	0.00 ± 0.00	0.73 ± 0.07 a	0.00 ± 0.00	0.97 ± 0.01 c
JB	0	8.44 ± 0.36 a	0.32 ± 0.09 a	0.25 ± 0.07 a	0.59 ± 0.05 a	0.00 ± 0.00	1.12 ± 0.34 a	0.98 ± 0.21 a	2.17 ± 0.59 a
	1	4.33 ± 0.21 b	0.18 ± 0.04 a	0.26 ± 0.05 a	1.39 ± 0.11 b	0.00 ± 0.00	1.47 ± 0.48 a	1.98 ± 0.26 b	4.78 ± 0.22 b
	3	6.62 ± 0.17 c	1.72 ± 0.11 b	0.14 ± 0.02 a	1.54 ± 0.12 b	0.00 ± 0.00	2.98 ± 0.29 b	1.20 ± 0.18 b	3.02 ± 0.17 c
	5	8.37 ± 0.33 a	1.75 ± 0.12 b	0.54 ± 0.09 a	1.21 ± 0.01 b	0.00 ± 0.00	5.48 ± 0.46 c	1.37 ± 0.31 b	3.10 ± 0.04 c
	7	4.13 ± 1.16 b	2.37 ± 0.16 c	0.51 ± 0.26 a	1.24 ± 0.14 b	0.00 ± 0.00	5.79 ± 1.29 c	0.31 ± 0.11 c	2.27 ± 0.18 a
	9	3.70 ± 0.23 b	3.62 ± 0.99 d	0.81 ± 0.13 b	1.48 ± 0.18 b	0.00 ± 0.00	5.90 ± 1.21 c	0.27 ± 0.19 c	2.16 ± 0.01 a
	11	2.97 ± 0.03 d	3.51 ± 0.28 d	1.38 ± 0.06 c	2.76 ± 0.29 c	0.00 ± 0.00	6.16 ± 0.59 c	0.33 ± 0.11 c	2.71 ± 0.10 a
	13	2.66 ± 0.03 d	6.38 ± 0.56 e	3.49 ± 0.09 d	2.68 ± 0.16 c	0.00 ± 0.00	5.67 ± 0.04 c	0.18 ± 0.03 c	1.86 ± 0.01 a
PCJ	0	3.10 ± 0.09 a	0.74 ± 0.12 a	0.00 ± 0.00	0.42 ± 0.04 a	0.00 ± 0.00	1.34 ± 0.04 a	0.00 ± 0.00	1.05 ± 0.04 a
	1	3.19 ± 0.04 a	0.82 ± 0.16 a	0.00 ± 0.00	0.10 ± 0.06 a	0.00 ± 0.00	1.69 ± 0.10 a	0.00 ± 0.00	0.50 ± 0.05 b
	3	1.47 ± 0.06 b	2.48 ± 0.21 b	0.00 ± 0.00	0.63 ± 0.05 a	0.00 ± 0.00	3.20 ± 0.19 b	0.00 ± 0.00	0.63 ± 0.07 b
	5	4.22 ± 0.11 c	4.01 ± 0.43 c	0.00 ± 0.00	2.99 ± 0.07 b	0.00 ± 0.00	5.70 ± 0.14 c	0.00 ± 0.00	1.41 ± 0.11 a
	7	2.29 ± 0.01 b	5.74 ± 0.60 d	2.78 ± 0.10 a	2.95 ± 0.11 b	0.00 ± 0.00	6.01 ± 0.05 c	0.00 ± 0.00	0.66 ± 0.02 b
	9	1.13 ± 0.14 b	7.26 ± 1.04 e	17.66 ± 0.19 b	3.06 ± 0.22 b	0.00 ± 0.00	9.12 ± 0.20 d	0.00 ± 0.00	0.86 ± 0.07 b
	11	2.12 ± 0.10 b	5.15 ± 1.09 d	25.15 ± 0.73 c	4.74 ± 0.19 c	0.00 ± 0.00	12.21 ± 0.06 e	0.00 ± 0.00	0.56 ± 0.12 b
	13	0.98 ± 0.00 d	6.28 ± 1.02 e	28.02 ± 0.16 c	8.69 ± 0.29 d	0.00 ± 0.00	15.89 ± 0.31 f	0.00 ± 0.00	0.50 ± 0.01 b
BCV	0	5.87 ± 0.18 a	0.36 ± 0.06 a	0.23 ± 0.04 a	0.41 ± 0.03 a	0.00 ± 0.00	1.96 ± 0.22 a	0.69 ± 0.07 a	2.20 ± 0.16 a
	1	5.91 ± 0.14 a	0.64 ± 0.05 a	0.24 ± 0.07 a	0.18 ± 0.01 a	0.00 ± 0.00	1.92 ± 0.28 a	1.26 ± 0.12 b	2.36 ± 0.15 a
	3	7.07 ± 0.52 b	3.11 ± 0.23 b	0.32 ± 0.03 a	0.26 ± 0.02 a	0.00 ± 0.00	2.24 ± 0.18 a	1.05 ± 0.13 b	2.30 ± 0.21 a
	5	8.36 ± 0.24 c	3.88 ± 0.28 b	0.00 ± 0.00	1.32 ± 0.03 b	0.00 ± 0.00	6.38 ± 0.24 b	0.76 ± 0.05 a	1.44 ± 0.09 b
	7	9.56 ± 0.76 c	6.03 ± 1.03 c	0.00 ± 0.00	0.45 ± 0.01 a	0.00 ± 0.00	4.63 ± 0.11 c	0.45 ± 0.17 a	3.73 ± 0.92 c
	9	7.11 ± 0.68 b	6.88 ± 1.11 c	0.00 ± 0.00	0.10 ± 0.00 c	0.00 ± 0.00	4.17 ± 0.04 c	1.74 ± 0.41 c	2.93 ± 0.30 a
	11	7.94 ± 0.04 b	6.77 ± 1.68 c	0.00 ± 0.00	0.09 ± 0.01 c	0.00 ± 0.00	4.83 ± 0.13 c	1.00 ± 0.03 b	3.64 ± 0.21 c
	13	3.15 ± 0.16 a	9.88 ± 0.36 d	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	3.52 ± 0.05 c	0.89 ± 0.33 b	2.42 ± 0.16 a

^a–f different letters in the same column display statistically different means (p < 0.05) between storage days for each burger sample; CON—burger without extracts; JB—burger with 2% of 1:2 common juniper by-product/blackberry leaves extract; PCJ—burger with 2% of 1:1 Padina pavonica/prickly juniper needles extract; BCV—burger with 2% of 2:1:1 blackberry leaves extract/catechin:vanillic acid.

. The biogenic amine profile varied among the different fish burgers. Importantly, no histamine accumulation was observed in any of the fish burgers, which is a positive indicator of food safety. Histamine is usually used as an indicator of harmful decomposition; however, sea bass and hake used for burger production have low histidine levels, which is a precursor amino acid for histamine formation. On the other hand, tryptamine, putrescine, and cadaverine increased during storage. However, the BCV burger showed the lowest levels of putrescine and cadaverine at the end of storage, while the PCJ burger exhibited the highest tyramine values. The results showed growth of LAB counts in PCJ burgers (Figure 6). This suggests that the LAB species present in PCJ burgers may be amine producers, or that compounds in PJ and BCV actively suppressed amine-producing bacteria.

In vacuum-packed sea bass stored at 2 °C, cadaverine and putrescine were found to be the dominant amines that accumulate [52]. Plant extracts that are rich in phenols are known to reduce biogenic amine formation by inhibiting decarboxylase enzymes and were found to be more effective in seafood products than essential oils [9,53]. Depending on the applied concentration of the extract, they can reduce the formation of putrescine, cadaverine, or even histamine in comparison to the untreated sample [9,54].

3.2. The Results of Microbiological Analyses

Fish is a commodity with a very short shelf life. Among others, its overall quality is influenced by storage temperature, packaging, and the initial quality of the raw material. The results of microbiological analyses of vacuum-packed fresh burgers are shown in Figure 6a–e. The initial TVC number reported for sea bass or hake is usually higher in the range of 3–4 log CFU/g [39,55,56]. The MEZ counts increased from 2.03 to 4.65 log CFU/g in the control burger. The initial MEZ count was lower in fish burgers with extracts; however, the count increased in all fish burgers throughout the storage. The lowest MEZ count at the end of the storage period was recorded for the JB burger (4.32 log CFU/g), followed by the BCV burger (4.49 log CFU/g). The PSI counts increased from 1.99 to 4.71 log CFU/g in the control burger during the 13 days (Figure 6a). All burgers with extracts had lower PSI microbial counts on the first day of storage, with 1.76 log CFU/g for JB, 1.79 log CFU/g for PCJ, and 1.91 log CFU/g for BCV. Total PSI counts increased with storage time in all fish burgers; however, JB and BCV burgers had lower PSI counts than the control burger at the end of the storage period (Figure 6b). Furthermore, the highest initial count of Enterobacteriaceae was found in the control burger (1.44 log CFU/g). Fish burgers with extracts had lower initial Enterobacteriaceae counts but slightly higher counts at the end of the storage period (Figure 6c). The counts of LAB differed significantly. Over the storage period, the LAB count was the highest in the PCJ burger, reaching 4.07 log CFU/g at the end (Figure 6d). Pseudomonas sp. counts increased from 1.87 to 4.88 log CFU/g in the control burger during the 19 days of shelf life in refrigerated conditions. All fish burgers with extracts had lower initial and final Pseudomonas sp. counts, with the lowest final count of 4.09 log CFU/g recorded for the JB burger (Figure 6e).

Microbiological standards and guidelines accept a total aerobic psychotropic count of 6 log CFU/g for chilled fish as acceptable quality for human consumption, while at microbial loads of 7–8 log CFU/g, the spoilage can be detected organoleptically [57,58]. Testing the effect of the plant extract mixtures (blackberry, juniper, Padina pavonica) alone and with pure compounds to evaluate their effects against microbial counts in fish products is limited to one previously published report. Previously studied extracts have been proven to be effective against Gram-positive pathogens, Pseudomonas and Shewanella spp. [16,27], and these results were tested in a study on sea bass fillets under MAP. Padina pavonica extract (alone or in combination) suppressed the bacterial growth, contributing to a shelf-life prolongation of the product [16]. Juniperus essential oil (0.3–0.6%) significantly inhibited total mesophilic bacteria count in vacuum-packed rainbow trout fillets stored at 2 ± 1 °C. The threshold of 7 log CFU/g was reached after 19 days in the control samples, and after 25 and 31 days in 0.3 and 0.6% juniper essential oil samples, respectively [46]. Rosemary was found to be effective in reducing microbial growth in sea bass fillets during storage at 4 °C and in extending the shelf life from 20 to 29 days [55]. Ozogul et al. [39] found that combined effect of plant-based extracts of blueberry and seaweeds, together with LAB supernatant, can increase the microbiological stability of modified packed sea bass fillets.

3.3. Fatty Acid Composition

The composition of fatty acids was determined at the beginning and end of the storage period. There is no significant difference in fatty acid composition between control burger and burgers with extracts on day 1 of refrigerated storage (

Table 3. Relative fatty acid composition (%) of fish burgers analyzed on days 1 and 11 of storage.

Treatment Group	CON		JB		PCJ		BCV
Storage Time (Days)	1	11	1	11	1	11	1	11
Fatty Acids (%)
C6:0	0.20 ± 0.05 a	0.02 ± 0.02 b	0.20 ± 0.04 a	0.03 ± 0.03	0.15 ± 0.15 c	0.15 ± 0.10 c	0.13 ± 0.06 c	0.08 ± 0.08 b
C8:0	0.01 ± 0.01 a	n.d.	0.04 ± 0.01 a	0.02 ± 0.02 a	0.01 ± 0.01 a	0.08 ± 0.06 a	0.01 ± 0.01 a	n.d.
C10:0	n.d.	n.d.	0.01 ± 0.00 a	n.d.	n.d.	0.02 ± 0.01 a	n.d.	n.d.
C11:0	n.d.	n.d.	n.d.	n.d.	n.d.	0.01 ± 0.01	n.d.	n.d.
C12:0	0.03 ± 0.00 a	0.02 ± 0.00 a	0.03 ± 0.00 a	0.02 ± 0.00 a	0.03 ± 0.00 a	0.03 ± 0.01 a	0.02 ± 0.00 a	0.03 ± 0.00 a
C13:0	0.01 ± 0.00 a	n.d.	0.01 ± 0.00 a	0.01 ± 0.01 a	0.01 ± 0.00 a	0.01 ± 0.00 a	n.d.	0.01 ± 0.00 a
C14:0	1.60 ± 0.19 a	1.64 ± 0.09 a	1.55 ± 0.06 a	1.66 ± 0.21 a	1.66 ± 0.13 a	1.77 ± 0.17 b	1.55 ± 0.03 a	1.70 ± 0.12 b
C14:1	0.04 ± 0.01 a	0.04 ± 0.01 a	0.04 ± 0.01 a	0.06 ± 0.03 a	0.04 ± 0.00 a	0.03 ± 0.01 a	0.04 ± 0.00 a	0.04 ± 0.00 a
C15:0	0.19 ± 0.00 a	0.18 ± 0.02 a	0.19 ± 0.01 a	0.20 ± 0.04 a	0.20 ± 0.01 a	0.24 ± 0.04 b	0.18 ± 0.02 a	0.19 ± 0.01 a
C15:1	n.d.	0.01 ± 0.00 a	0.01 ± 0.00 a	0.01 ± 0.01 a	n.d.	0.01 ± 0.01 a	n.d.	0.01 ± 0.01 a
C16:0	15.42 ± 0.65 a	15.63 ± 0.31 a	15.07 ± 0.14 a	15.46 ± 0.57 a	15.59 ± 0.37 a	15.61 ± 0.46 a	15.38 ± 0.14 a	15.45 ± 0.41 a
C16:1	2.88 ± 0.09 a	2.84 ± 0.08 a	2.75 ± 0.03 a	2.83 ± 0.21 a	2.86 ± 0.05 a	2.92 ± 0.23 b	2.76 ± 0.04 a	3.10 ± 0.11 b
C17:0	0.17 ± 0.01 a	0.18 ± 0.00 a	0.16 ± 0.00 a	0.21 ± 0.05 b	0.18 ± 0.01 a	0.17 ± 0.00 a	0.18 ± 0.00 a	0.17 ± 0.00 a
C17:1	0.20 ± 0.02 a	0.22 ± 0.00 a	0.07 ± 0.12 b	0.21 ± 0.01 a	0.19 ± 0.00 a	0.13 ± 0.11 c	0.21 ± 0.01 a	0.15 ± 0.06 c
C18:0	4.50 ± 0.07 a	4.52 ± 0.14 a	4.30 ± 0.08 a	4.69 ± 0.23 b	4.47 ± 0.13 a	4.86 ± 0.04 b	4.32 ± 0.12 a	4.42 ± 0.03 a
C18:1 n-9t	0.07 ± 0.02 a	0.11 ± 0.06 b	0.12 ± 0.08 b	0.10 ± 0.01 a	0.11 ± 0.08 a	0.07 ± 0.01 a	0.14 ± 0.08 b	0.06 ± 0.03 a
C18:1 n-9c	36.80 ± 0.25 a	36.88 ± 0.37	36.75 ± 0.13	36.67 ± 0.61	36.91 ± 0.29	36.51 ± 0.38	36.97 ± 0.20	36.41 ± 0.27
C18:2 n-6t	0.03 ± 0.02 a	0.03 ± 0.01 a	0.01 ± 0.00 a	0.02 ± 0.01 a	0.01 ± 0.00 a	0.01 ± 0.00 a	n.d.	n.d.
C18:2 n-6t	25.80 ± 0.26 a	25.50 ± 0.16 a	25.65 ± 0.23 a	25.32 ± 0.26 a	25.62 ± 0.25 a	25.53 ± 0.29 a	25.56 ± 0.23 a	25.75 ± 0.13 a
C18:3 n-6	0.36 ± 0.01 a	0.38 ± 0.01 a	0.38 ± 0.01 a	0.40 ± 0.03 a	0.36 ± 0.01 a	0.37 ± 0.01 a	0.38 ± 0.01 a	0.36 ± 0.01 a
C18:3 n-3	3.90 ± 0.07 a	3.72 ± 0.17 a	3.89 ± 0.10 a	3.85 ± 0.05 a	3.86 ± 0.08 a	3.90 ± 0.12 a	3.84 ± 0.04 a	4.01 ± 0.08 b
C20:0	n.d.	0.03 ± 0.03 a	n.d.	0.07 ± 0.03 a	0.04 ± 0.04 a	n.d.	0.02 ± 0.02 a	0.03 ± 0.03 a
C20:1	2.00 ± 0.13 a	2.05 ± 0.05 a	2.11 ± 0.15 a	2.01 ± 0.18 a	1.98 ± 0.08 a	2.01 ± 0.09 a	2.15 ± 0.01 a	1.87 ± 0.08 b
C20:2	1.15 ± 0.05 a	1.20 ± 0.06 a	1.22 ± 0.04 a	1.14 ± 0.08 a	1.19 ± 0.07 a	1.14 ± 0.04 a	1.15 ± 0.03 a	1.20 ± 0.02 a
C20:3	0.56 ± 0.03 a	0.54 ± 0.02 a	0.64 ± 0.05 b	0.53 ± 0.01 a	0.56 ± 0.03 a	0.51 ± 0.04 a	0.58 ± 0.04 a	0.55 ± 0.03 a
C20:3 n-6	0.15 ± 0.00 a	0.16 ± 0.05 a	0.21 ± 0.06 b	0.19 ± 0.10 b	0.14 ± 0.00 a	0.14 ± 0.01 a	0.15 ± 0.03 a	0.16 ± 0.02 a
C20:4 n-6	0.30 ± 0.01 a	0.33 ± 0.11 a	0.37 ± 0.06 b	0.33 ± 0.08 a	0.29 ± 0.01 a	0.28 ± 0.01 a	0.32 ± 0.01 a	0.32 ± 0.04 a
C20:5n-3 (EPA)	1.27 ± 0.03 a	1.04 ± 0.03 b	1.37 ± 0.13 c	1.34 ± 0.03 c	1.22 ± 0.03 d	1.21 ± 0.04 d	1.32 ± 0.02 a	1.28 ± 0.08 a
C22:0	0.21 ± 0.03 a	0.18 ± 0.04 a	0.23 ± 0.04 a	0.14 ± 0.04 b	0.26 ± 0.13 c	0.21 ± 0.01 a	0.18 ± 0.05 a	0.21 ± 0.02 a
C22:1 n-9	0.34 ± 0.02 a	0.33 ± 0.04 a	0.42 ± 0.06 b	0.36 ± 0.08 a	0.33 ± 0.03 a	0.32 ± 0.02 a	n.d.	0.34 ± 0.04 a
C22:2	n.d.	0.31 ± 0.03 a	0.30 ± 0.05 a	0.34 ± 0.11 a	0.10 ± 0.14 b	0.20 ± 0.16 c	0.29 ± 0.02 a	0.30 ± 0.03 a
C22:6n-3 (DHA)	1.56 ± 0.11 a	1.23 ± 0.01 b	1.61 ± 0.02 a	1.58 ± 0.09 a	1.44 ± 0.03 c	1.36 ± 0.02 c	1.57 ± 0.02 a	1.52 ± 0.02 a
C24:1	0.23 ± 0.03 a	0.20 ± 0.02 a	0.26 ± 0.02 a	0.19 ± 0.02 b	0.21 ± 0.02 a	0.20 ± 0.03 b	0.23 ± 0.02 a	0.26 ± 0.06 a
∑ SFA	22.34 ± 1.01 a	22.43 ± 0.67 a	21.79 ± 0.37 a	22.50 ± 1.22 b	22.58 ± 0.99 b	23.16 ± 0.91 b	21.99 ± 0.46 a	22.30 ± 0.72 b
∑ MUFA	42.56 ± 0.57 a	42.69 ± 0.65 a	42.53 ± 0.61 a	42.46 ± 1.16 a	42.64 ± 0.56 a	42.19 ± 0.87 a	42.50 ± 0.35 a	42.26 ± 0.66 a
∑ PUFA	35.09 ± 0.59 a	34.46 ± 0.72 a	35.64 ± 0.76 a	35.04 ± 0.86 a	34.78 ± 0.65 a	34.66 ± 0.83 a	35.16 ± 0.45 a	35.42 ± 0.57 a
EPA	1.27 ± 0.03 a	1.04 ± 0.03 b	1.37 ± 0.13 c	1.34 ± 0.03 c	1.22 ± 0.03 d	1.21 ± 0.04 d	1.32 ± 0.02 c	1.28 ± 0.08 c
DHA	1.56 ± 0.11 a	1.23 ± 0.06 b	1.61 ± 0.02 c	1.58 ± 0.09 ac	1.44 ± 0.03 d	1.36 ± 0.12 e	1.57 ± 0.02 a	1.52 ± 0.12 a
EPA+DHA	2.83 ± 0.15 a	2.72 ± 0.09 b	2.98 ± 0.16 c	2.92 ± 0.12 c	2.66 ± 0.06 d	2.58 ± 0.16 d	2.89 ± 0.05 a	2.80 ± 0.20 ab
n-3	7.39 ± 0.23 a	6.70 ± 0.38	7.62 ± 0.32	7.50 ± 0.28	7.17 ± 0.16	7.12 ± 0.30	7.42 ± 0.10	7.48 ± 0.33
n-6	32.25 ± 0.45 a	32.19 ± 0.63	32.66 ± 0.60	32.12 ± 0.73	32.12 ± 0.59	32.08 ± 0.67	32.27 ± 0.41	32.65 ± 0.37
n-3/n-6 *	0.23	0.21	0.23	0.23	0.22	0.22	0.23	0.23
PUFA/SFA *	1.57	1.53	1.64	1.56	1.54	1.50	1.6	1.59

n.d.—not detected, * calculated based on the means; ^a–e different letters in the same row display statistically different means (p < 0.05) between treatment groups and storage times; SFA—saturated fatty acids; MUFA—monounsaturated fatty acids; PUFA—polyunsaturated fatty acids; EPA—eicosapentaenoic acid; DHA—docosahexaenoic acid; CON—burger without extracts; JB—burger with 2% of 1:2 common juniper by-product/blackberry leaves extract; PCJ—burger with 2% of 1:1 Padina pavonica/prickly juniper needles extract; BCV—burger with 2% of 2:1:1 blackberry leaves extract/catechin/vanillic acid.

). The sum of saturated fatty acids ranged from 21.79 ± 0.37 to 22.58 ± 0.99% for all burgers at the beginning of the storage period and from 22.23 ± 0.67 to 23.16 ± 0.91% at the end of the storage time, dominated by palmitic (C16:0) and stearic (C18:0) fatty acids. The sum of monounsaturated fatty acids (MUFA) ranged from 42.2 to 42.7% throughout the storage, dominated by oleic acid (C18:1cis9). The sum of polyunsaturated fatty acids (PUFA) varied only slightly through the shelf-life period. In the control sample, PUFA decreased by 2%, the sum of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by 4%. The PUFA in burgers with extracts and pure compounds did not change during storage.

4. Conclusions

To summarize, the three studied mixtures of extracts—common juniper by-product and blackberry leaves, P. pavonica and prickly juniper needles, blackberry leaves and catechin and vanillic acid—were evaluated for their potential to increase the chemical and microbial quality of vacuum-packed fish burgers. All of these extracts were previously proven to have good antioxidant and antimicrobial activity [16,27,28]. In the fish burger model, extracts demonstrated an inhibitory effect on lipid oxidation, especially in PCJ and BCV. Similarly, they have proven the inhibitory effect on volatile amine formation (both TVB-N and TMA), particularly in BCV samples. This effect is probably a result of the high concentration of phenolic compounds in the extracts. In JB and PCJ, the number of Pseudomonas sp. was lower than in the control, while MEZ and PSI were not affected. The overall biogenic amine accumulation was low and not affected by the extracts. The LAB counts, putrescine, and tyramine increased only in PCJ samples. The studied extracts offer a promising natural strategy to improve lipid oxidation in fresh fish burgers during chilled storage. Further research should focus on optimizing these bio-preservatives (e.g., identifying the most active compounds, the mechanism of action for particular compounds, more precise concentrations, and the effect of combining different extracts and phenolics) and accurately predicting their effects on a broader spectrum of parameters over longer storage in different food products.