*Article* **Combined Effects of Parsnip Fermented Juice and Hawthorn Extract Regarding Pork Mince Stability: Physico-Chemical and Microbiological Aspects**

**Corina Nicoleta Predescu 1,\*, Camelia Papuc 1,2, Georgeta Stefan 1, Bogdan Tas,bac 1, Georgeta Temocico 3, Mihaela Sărăcilă 4 and Arabela Elena Untea 4**

**Abstract:** Parsnip fermented juice (PFJ) and hawthorn extract (HE) were identified as natural nitrite and antioxidant sources for pork mince. This study aimed to determine the effects of varying levels of HE added to a constant concentration of PFJ on lipids stability, heme pigment conversion degree, residual nitrite content, and spoilage bacteria growth, during refrigeration, compared with the combined effect of synthetic nitrite and sodium ascorbate (SA). Pork mince was formulated in six different ways with sterile distilled water (NC), 100 ppm synthetic nitrite and 50 ppm SA (PC), PFJ in the concentration of 100 ppm NO2 − (T1), constant level of PFJ (100 ppm NO2 −), and increased level of HE, 50, 25 and 10 ppm GAE (T2, T3 and T4). During the experiment, pH increased for all the treatments, but the addition of PFJ alone or in combination with HE, it was maintained below the NC pH value. The lowest TBARS values and the highest PUFA concentrations were found in the T3, T4, and PC treatments. Of all the samples, the lowest residual nitrite values were found for T2. The highest NO-heme values were found for T2 and PC. After 9 days of storage, TVC results were higher than 5.69 logs CFU/g for all treatments. Overall, the obtained results showed that the combination of HE and PFJ could be a promising natural preservative for minced meat that could replace synthetic preservatives.

**Keywords:** parsnip fermented juice; hawthorn extract; natural nitrite; natural antioxidant; lipids stability; spoilage bacteria; heme pigment conversion degree; bioactive compounds

#### **1. Introduction**

Oxidative processes and meat spoilage bacteria are the most important factors in decreasing the shelf life of meat and meat products. Meat oxidation starts during slaughtering and continues in post-slaughtering conditions, handling, processing, and storage [1,2]. Meat is susceptible to spoilage bacteria due to its favourable growth conditions, high water content, nitrogen-containing molecules, carbohydrates, lipids, lactic acid, vitamins, and minerals. These degradative processes result in the development of off-odours, off-flavour, off-taste, loss of colour, loss in nutritional value, slim formation, and toxic compounds generation, making the meat undesirable for human consumption [3–5]. In the meat industry, synthetic antioxidants and nitrates and nitrites inhibit oxidative processes, bacterial growth, and input attractive colour. Lipid oxidation may be slowed down by synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), propyl gallate (PG), and sodium ascorbate and ascorbate.

**Citation:** Predescu, C.N.; Papuc, C.; Stefan, G.; Tas,bac, B.; Temocico, G.; S ˘ar ˘acil ˘a, M.; Untea, A.E. Combined Effects of Parsnip Fermented Juice and Hawthorn Extract Regarding Pork Mince Stability: Physico-Chemical and Microbiological Aspects. *Agriculture* **2023**, *13*, 432. https://doi.org/ 10.3390/agriculture13020432

Academic Editor: Javier Álvarez-Rodríguez

Received: 29 December 2022 Revised: 4 February 2023 Accepted: 10 February 2023 Published: 12 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

To inhibit the grow spoilage bacteria and improve meat colour, synthetic additives, such as potassium/sodium nitrite and potassium/sodium nitrate are used. According to commission regulation (EU) No 1129/2011, the amount of nitrite permitted for use as a food additive in cured meat is currently 150 mg kg−1. Many studies have suggested the toxicity and carcinogenicity of synthetic antioxidants with phenolic structures [6–8] and synthetic nitrites and nitrates [9,10] and for this reason, the researchers interested in replacing these synthetic substances with natural ones has increased in recent decades [11–16]. Many research studies have demonstrated the antioxidant and antimicrobial activity of plant extracts rich in phenolic compounds [14]. Vegetables are important sources of natural nitrates, but nitrate concentrations vary widely among plants, plant parts, and growing conditions. During the fermentation process, nitrates from plant juices can be converted into nitrites and, after concentration, concentrated fermented vegetable plant juices or fermented plant powders rich in natural nitrites result. The high nitrate content in the fermented vegetable juice/powder led researchers to study it as a nitrite natural source for the meat industry [17]. In recent years, consumer preferences have shifted towards requiring health-friendly, high-quality, nutrient-rich natural products. Parsnip fermented juice appears to be highly compatible with processed meat products because it has a high content of nitrates, very little vegetable pigment, and a mild, pleasant flavour profile. On the other hand, extracts of hawthorn berries are sources of natural antioxidants with previously proven preservative effects for meat products [5]. In fact, the present research highlights the preservative activity of the active compounds in hawthorn and nitrite-rich fermented juice. This study emphasizes the effects of varying levels of hawthorn berries ethanolic extract (HE) added to a constant concentration of parsnip fermented juice (PFJ) on lipids stability, heme pigment conversion degree, residual nitrite content, and spoilage bacteria growth in pork mince during refrigeration, compared to the combined effect of synthetic nitrite and sodium ascorbate. This study aimed to evaluate the combined effects of parsnip fermented juice and hawthorn phenolics on lipids and colour stability, and on spoilage bacteria growth in pork mince.

#### **2. Materials and Methods**

#### *2.1. Parsnip Fermented Juice (PFJ) Obtaining*

Parsnip roots were bought from a local market near Bucharest. The parsnip roots were cut into small pieces (1 × 1 cm) and then placed in a homogenizer with sterile distilled water in a ratio of 1:2 (*w*/*v*), and left to rest for 3 h at 4 ◦C. After homogenization, filtration and sterilization *Staphylococcus xylosus* (ATCC 29971) were added at 108 cfu/mL in a shaker incubator at 37 ◦C for 36 h [18]. Next, the mixture was filtered through Whatman No. 1 filter paper and evaporated using a rotary evaporator (Heidolph Laborota 4000). The concentrated fermented parsnip juice had a pH of 5.31 and 6237.5 ppm nitrite content. The nitrite concentration was assayed by the AOAC method [19]. The concentrated juice was kept in the refrigerator until used.

#### *2.2. Hawthorn Extract (HE) Obtaining*

The dried hawthorn fruits were collected from the forests near Câmpulung Muscel, Arges, county. After grinding, using a kitchen milling machine, the obtained powder was mixed with 60% ethanol (*v*/*v*), in an extraction rapport of 1:10 (*w*/*v*). Then was vigorously mixed and left to stand for 5 h. After that, the extraction was continued in a water bath (GFL 1092) at 60 ◦C for 3 h. The mixture was filtered through Whatman No. 1 filter paper and concentrated with a rotary evaporator (Heidolph Laborota 4000) to a temperature lower than 80 ◦C. The total phenolic content was assayed using Folin Ciocalteu reagent [20] and was expressed as mg gallic acid equivalent/mL (mg GAE/mL).

#### *2.3. Experimental Setup and Preparation of Meat Samples*

Fresh pork leg was purchased from a local butcher. After washing with distilled sterile water, all subcutaneous and intramuscular fat and visible connective tissue were removed with a knife. The meat was manually chopped into cubes of approximately 2 cm3 and then minced in a grinder equipped with a 10 mm plate, followed by grinding through a 3.5 mm plate. The meat was divided into 6 portions of one kilogram. The samples were treated with sodium nitrite (SN) and sodium ascorbate (SA), parsnip fermented juice (PFJ), and hawthorn extract (HE) at different concentrations, as shown in Table 1. Sodium ascorbate and sodium nitrite were analytical reagents and were obtained from Sigma-Aldrich, St Louis, MO, USA.

**Table 1.** Pork mince treatments with sodium nitrite (SN), sodium ascorbate (SA), parsnip fermented juice (PFJ), and hawthorn extract (HE).

Abbreviation: SN, sodium nitrite; SA, sodium ascorbate; PFJ, parsnip fermented juice; HE, hawthorn extract; NC, negative control; PC, positive control; GAE, gallic acid equivalent; ppm, parts per million and it expresses milligrams per kg (mg/kg); T1, treatment 1; T2, treatment 2; T3, treatment 3; T4, treatment 4.

For the pork-minced formulation, the ingredients (SN, SA, PFJ, HE) were dissolved in cold (4 ◦C) sterilized distilled water in a 100 mL final volume. For the negative control, 100 mL of sterilized distilled water was used. All meat samples were homogenized using a food processor (Moulinex DP 700), packed in plastic film, and stored at 4 ◦C for 9 days.

#### *2.4. Physicochemical Analysis*

#### pH Value

The pH value of all samples was measured using a pH meter (Hanna Instruments, Cluj Napoca, Romania) by direct measurement with a glass electrode calibrated with the phosphate buffers 4.0 and 7.0 at room temperature (21 ◦C). Ten grams of sample was homogenized with distilled water in a ratio of 1:100 (*w*/*v*) for 30 min. After filtration, the pH of the filtrate was measured [21].

#### *2.5. Chemical Analysis*

2.5.1. Chromatographic Profile of Fatty Acids

Lipid Extraction and Fatty Acid Methyl Esters

The fatty acids profile of lipids from minced meat was determined as fatty acids methyl esters (FAME). Lipid extractions were made according to [22] method and FAME was prepared by transmethylation using 2 M KOH in methanol and normal heptane according to the method described by [23].

#### Gas-chromatographic Analysis of Fatty Acid Methyl Esters

Fatty acid methyl esters (FAME) were quantified by gas chromatography (GC) using a Perkin-Elmer-Clarus 500 system with a flame ionisation detector (FID), capillary column injection system and a silica capillary column SGE (BPX70, 60 m; 0.25 mm inner diameter, 0.25 μm film, Agilent). Gas-chromatographic conditions were according to the procedure described by [5]. Each fatty acid was expressed as g/100 g fatty acid methyl esters (FAME).

#### 2.5.2. Thiobarbituric Acid Reactive Substances (TBARS) Value

Lipid oxidation in the minced pork was monitored by measuring thiobarbituric acid reactive substances (TBARS) every 3 days during refrigeration storage. TBARS value was assayed by the method described by [24]. Briefly, 0.5 g minced meat was treated with 2.5 mL thiobarbituric acid solution. After homogenisation, the tube with the mixture was immersed in a boiling water bath for 10 min. After cooling under running tap water, sonication for 30 min, and centrifugation, the absorbance of the supernatant was read at 532 nm. A standard curve was prepared using 1,1,3,3-tetramethoxypropane and the concentration ranging from 0 to 10 ppm. TBARS value was expressed as mg of malondialdehyde equivalents/kg of the sample (mg MDA/kg).

2.5.3. Nitrosyl Hemochrome, Total Pigment Content, and the Heme Pigment Conversion Degree

Nitrosyl hemochrome and total pigments were measured after extraction with acetone and acidified acetone [25]. For quantification of the nitrosyl hemochrome, a 10 g sample was mixed with 43 mL of acetone-water solution (acetone: water, 40.5:2.5, *v*/*v*) in reduced light. After 5 min, the mixture was filtered through Whatman No. 1 paper, and the filtrate absorbance was measured at 540 nm using acetone water solution (80% acetone, 20% water) for blank. NO-heme pigment concentration was calculated using formula (1).

NO-heme concentration (ppm hematin acid) = sample A540 × 290 (1)

For total pigment measurement, a 10 g sample (65% water) was mixed with 40.4 mL acetone, 1.6 mL water and 1 mL of concentrated HCl. The mixture was stored in dark at the room temperature, stirring from time to time. After 1 h, the homogenates were filtrated and the absorbance of the filtrate was read at 640 nm against the same solvent mixture used for the homogenate preparation. The optical density read at 640 nm was multiplied by 680, according to formula (2), to express the total pigment concentration.

Total pigment concentration (ppm hematin acid) = A640 × 680 (2)

The heme pigment conversion degree was calculated using formula (3).

$$\% \text{Conversion} = \text{(NO-Herme/Total-Hence)} \times 100\tag{3}$$

#### 2.5.4. Residual Nitrite Level

Residual nitrite levels in the minced samples were assayed using the method recommended by [26]. For nitrite extraction, 5 g of sample was mixed completely with 40 mL hot water and quantitative transferred to a 500 mL volumetric flask. After the addition of about 300 mL, the volumetric flask was immersed in a shaking water bath for 2 h at 80 ◦C. Then, the mixture was cooled at room temperature, diluted to volume with water, mixed and filtrated. The colourimetric method using Griess diazotization was performed. Residual nitrite level was determined by comparison with the prepared standard curve and was expressed as ppm NO2 − (μg NO2 −/g sample).

#### 2.5.5. Volatile Basic Nitrogen (VBN) Value

To determine the extent of protein deterioration during refrigeration storage, volatile basic nitrogen value was performed by the method of [27]. Briefly, 5 g of sample were mixed with 30 mL of 5% (*w*/*v*) trichloroacetic acid (TCA). The homogenate was made up to 50 mL of final volume with 5% (*w*/*v*) TCA and filtered using the Whatman filter paper No. 1. One mL of filtrate and 1 mL of borate buffer were placed in the outer and inner Conway dish, respectively. After 100 min incubation at 37 ◦C, the inner solution was titrated with 0.01 N HCl and the titration volume was recorded. The results were expressed as mg%.

#### 2.5.6. Microbiological Analysis

Total viable count (TVC) was determined in plate count agar by the pour-plate method [28]. A total of 25 g of sample was aseptically weighed and homogenized with sterile 0.1% peptone water in the ratio 1:9 (*w*/*v*) for 1 min using a stomacher (400 circulators Seward Ltd.UK) at a speed of 6000 rpm. The homogenized sample was serially diluted (1:10) in sterile 0.1% peptone water. One mL sample of serial dilutions was plated into plate count agar and then incubated at 35–37 ◦C for 48 h. Microbiological data were expressed as the logarithm of the number of colony-forming units (logCFU/g). All counts were performed in triplicate.

#### *2.6. Statistical Analysis*

The effect of treatments on the fatty acids profile of minced pork was performed using one-way ANOVA (XLStat, Addinsoft, New York, NY, USA). The effect of treatments and storage time on pH value, TBARS, NO-heme, total heme, heme pigment conversion degree, residual nitrite level, volatile basic nitrogen, and the total viable count was performed using two-way ANOVA (XLStat, Addinsoft, New York, NY, USA). The Tuckey test was used to predict differences among the criteria; the effects were considered significant if *p* < 0.05. The statistical model included the fixed effects of treatments (NC, PC, T1, T2, T3, T4) and storage time (0, 3, 6, 9 days) and their interactions. For correlations between parameters, Pearson's correlation was used.

#### **3. Results and Discussions**

*3.1. Physico-Chemical Analysis*

pH Value

Several studies demonstrated that increased refrigeration storage increased raw meat's pH [29–31]. It was suggested that increases in pH during refrigeration are due to the volatile basic compounds, such as ammonia, methylamine, dimethylamine and trimethylamine, and microbial catabolites, which result in meat spoilage [4,32,33]. The pH variations for meat samples according to the storage time are shown in Table 2.

**Table 2.** Changes in pH values of pork mince depending on treatment and storage time.

a–d Means in a row without a common letter significantly differ. NC—negative control; PC—positive control; T1 –treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4– treatment with PFJ +HE, 10 ppm GAE.

On day 0, a slight difference was found between the positive control pH values and others, due to the residual acidity of hawthorn phenolics and PFJ. After 3 days of refrigeration, the pH increased for all samples, but the highest increase was found in the NC. The increase in the pH during storage was lower in the first 3 days and higher in the last 6 days. On the 6th day, T2 and T3 pH values were similar to PC. After 9 days of storage, the pH value of T1 was significantly lower than the pH value found in NC (*p* < 0.05). T2 pH value was significantly lower than the pH values found in T3 and T4 (*p* < 0.05). HE addition in pork mince inhibited pH values increasing in a relation depending on HE levels; thus, after 9 days of refrigeration storage, the pH values decreased in order T4, T3, and T2. By comparison of pH values for samples treated with PFJ + HE, it can be seen that samples treated with 25 and 10 ppm GAE (T3 and T4) resulted in a greater increase in pH (*p* < 0.05); formulation with 50 ppm GAE (T2) resulted in an increase in pH values similar to PC. The slight increase in the pH in the meat samples treated with the PFJ and PFJ + HE treatments (T1, T2, T3 and T4) is attributed to the inhibitory effect of antimicrobial compounds found both in HE and PFJ on the growth and proliferation of spoilage microorganisms that metabolize basic nitrogen compounds, such as amino acids, L-carnitine, lecithin, and choline [30,31].

#### *3.2. Chemical Analysis*

#### pH Value

Chromatographic Profile of Fatty Acids

Lipid deterioration is the major cause of the loss of bioavailability and sensory quality of the meat and is due to fatty acids oxidation and, to a lesser extent of fatty acids catabolism by spoilage bacteria. Lipid oxidation in muscle systems is initiated at the membrane level in the intracellular phospholipid fractions, with high polyunsaturated fatty acids (PUFA) content [34]. In minced meat, PUFA oxidation is the result of the interaction with endogenous prooxidants, such as metalloproteins [35], and with exogenous prooxidants, such as iron from grinding machines and singlet oxygen. The effects of PFJ and PFJ + HE, in concentrations 50, 25, and 10 mg GAE/kg, on the fatty acids profile, after 9 days of refrigeration are shown in Table 3.

**Table 3.** Fatty acid profile of pork mince with different treatments after 9 days of refrigeration.

1 FAME—fatty acids methyl esters. FA—fatty acids; SFA—saturated fatty acids; MUFA—monounsaturated fatty acids; PUFA—polyunsaturated fatty acids. a–f Means in a row without a common superscript letter significantly differ (*p* < 0.05). NC—negative control; PC—positive control; T1—treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4—treatment with PFJ +HE, 10 ppm GAE.

In all samples, the monounsaturated fatty acids (MUFA) showed the highest degree (44.61–48.63%), with the oleic acid (C18:1) being the most abundant fatty acid (44.58–44.79%), whereas polyunsaturated fatty acids (PUFA) showed the lowest percent (14.30–16.67%), with linoleic acid (C18:2n−6) the most abundant PUFA (12.21–13.80%). Saturated fatty acids (SFA) were found in the intermediate degree (35.04–40.98%), with palmitic (C16:0) and stearic (C18:0) acids the most abundant (23.35–24.35% and 10.13–10.61%, respectively). The results are in agreement with the results obtained by [36] that found a similar fatty acid profile in pork meat. Results show differences in fatty acid profile for all treatments, although some of them were statistically significant. For PFJ + HE treatments, (T2, T3 and T4), the total PUFA degree was significatively higher than the degree found in NC (*p* < 0.05), on the last day of determination. The highest total PUFA degree was found in PC

samples, treated with sodium nitrite and sodium ascorbate (PC). The total PUFA degree in sample T3, treated with PFJ + HE, 25 ppm GAE, follows an increasing path as PC (*p* < 0.05). Except for the formulation with PFJ + HE 50 ppm GAE/kg (T2), the other treatments with PFJ + HE (T3 and T4), significantly (*p* < 0.05) increased the stability of unsaturated fatty acids to oxidation, compared with samples formulated with PFJ alone (T1). The lowest degree for total PUFA was found for T1. Comparing the PUFA results obtained for T2, T3, and T4, a prooxidant effect on minced pork lipids by HE added at 50 ppm GAE/kg can be suggested. According to the results from the present study, the concentrations of 25 and 10 mg GAE/kg, seem to protect the essential fatty acids, linoleic (C18:2n−6), linolenic (C18:3n−3), and arachidonic (C20:4n−6) acids against oxidative damage, comparatively with T1. Some authors have shown that colonial-type salami treated with rosemary extract alone or in association with celery powder had a higher PUFA content compared with control samples. [37]. Some authors used Mediterranean berries' ethanolic extracts as antioxidants in pork burgers subject to cooking and chilled storage and found that the protective effect of ethanolic extracts on PUFA decreased in the following order: *Rosa canina*, *Rubus ulmifolius*, *Arbusto unedo*, and *Crataegus monogyna*. The highest level of linoleic acid they found in patties treated with the ethanolic extract obtained from *Arbusto unedo* berries [38].

#### *3.3. Thiobarbituric Acid Reactive Substances (TBARS) Value*

Generally, lipid oxidation affects bioavailability and sensory quality in minced meat. A good indicator of lipid oxidation level is the TBARS value. It was suggested that the TBARS value at 0.5 mg/kg was a threshold value for rancidity perception by consumers [39]. The results regarding the changes in TBARS values for the six evaluated treatments are presented in Table 4.

**Table 4.** Changes in thiobarbituric acid reactive substances values of minced pork depending on treatment and storage time.

a–e Means in a row without a common letter significantly differ. NC—negative control; PC—positive control; T1 –treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4– treatment with PFJ +HE, 10 ppm GAE.

The analysis of variance for the TBARS data indicates that the TBARS values for pork mince were influenced by the refrigeration period (*p* < 0.001) and treatments. Overall, TBARS values increased during refrigeration storage, but the lowest TBARS values were found in PC pork mince (*p* ≤ 0.05). The sample treated with PFJ alone (T1) had lower TBARS values than NC, but higher than PC (*p* ≤ 0.05). The addition of HE in the minced pork affects the TBARS values in a concentration-dependent manner, after 3 days of refrigeration. The best antioxidant activity was found in the sample treated with PFJ + HE in a concentration equal to 25 ppm GAE (T3), followed by the samples treated with PFJ + 10 ppm GAE HE (T4), on the 6th day of the determination. On the last day of the investigation (9th day), the samples treated with PFJ + 50 ppm HE (T2) showed the highest TBARS values, comparatively with T3 and T4. The result obtained showed that the capacity of PFJ + HE to inhibit lipid oxidation in minced pork depends on the amount of HE used. HE in concentrations of 50 ppm GAE/kg meat has prooxidant activity, but HE in a concentration of 25 and 10 mg GAE/kg influenced the antioxidant activity as the association of sodium nitrite with sodium ascorbate (PC).

Another study showed the TBARS values after the preparation of beef patties using resveratrol as an antioxidant in concentrations as 110 μmol/kg meat and 550 μmol/kg meat, during 9 days of refrigeration. The highest TBARS values were found in samples treated with 550 μmol/kg meat, comparatively with TBARS values found in samples treated with 110 μmol/kg meat, but both were lower than those found in control samples [40]. It was reported different TBARS values for cooked pork patties, prepared with ethanolic berry extracts, and stored by refrigeration than those found in control samples (1.071 mg MDA/kg) [38]. So, higher TBARS values were reported for *Crataegus monogyna* (0.181 mg MDA/kg) and *Rosa canina* (0.143 mg MDA/kg) treatment extracts, but lower TBARS values for *Rubus ulmifolius* (0.082 mg MDA/kg) and *Arbusto unedo* (0.113 mg MDA/kg) treatment extracts.

#### *3.4. Nitrosyl Hemochrome, Total Pigment Content, and the Heme Pigment Conversion Degree*

Nitrosyl hemochrome (NO-heme), the pink pigment of cured meat, is formed mainly from NO(II)Mb resulting in the reaction of myoglobin (Mb) with NO generated from nitrite conversion in the presence of ascorbate [41]. Some studies suggested that the addition of ascorbate in the muscle reduces met(III)Mb to deoxy(II)Mb and the simultaneously generated NO from nitrite binds to deoxy(II)Mb or can reduce nitrosylate met(III)Mb under anaerobic conditions, resulting in the formation of NO(II)Mb [41].

The studies regarding the effect of pH on the stability of nitrosyl hemochromogen suggest that NO-Heme is extremely unstable at weakly acid pH, and this explains why pH affects the apparent colour of cured meat products with storage time [42]. Table 5 showed the results obtained for nitrosyl hemochrome (NO-Heme) values of minced pork formulated with parsnip fermented juice (PFJ) alone, parsnip fermented juice + hawthorn extract (PFJ + HE) compared to untreated minced pork (NC), and synthetic added compounds (PC).

**Table 5.** Changes in NO-heme value, total heme value, and heme pigment conversion degree of minced pork depending on treatment and storage time.

#### **Table 5.** *Cont.*

a–e Means in a row without a common letter significantly differ. NC—negative control; PC—positive control; T1–treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4–treatment with PFJ +HE, 10 ppm GAE

The highest value for NO-Heme during 9 days of refrigeration was found in PC. In the sample formulated only with PFJ (T1), NO-Heme content was significantly lower, compared with PC and samples formulated with PFJ + HE (T2, T3 and T4) (*p* < 0.05). The formulation of the minced meat with PFJ and HE increased NO-Heme content, in a relation dependent on the HE level in samples (Table 5).

After 9 days of refrigeration storage, in the samples treated with PFJ + HE, the content of NO-Heme increased in the order: T2, T3, and T4. Obtained results showed that HE accelerates the heme pigments to react with NO in a concentration-dependent manner. The nitrosylation reaction stimulated by HE and SA may be due to the lowering of the pH values on day 0 and of the reaction between phenolics and nitrite [43]. It was reported that a pH decreasing only 0.2 unit, doubles the rate of colour formation through nitrite-myoglobin reaction [44]. It was suggested that the final colour properties of nitrite also increase with decreasing pH and this constitutes the basis for product formulation with acidulants to accelerate curing [44,45].

Table 5 presents the results regarding total heme pigment values obtained for all treatments during 9 days of refrigeration. In all samples, a slight decrease in total heme pigment was found after 9 days of storage. Significant differences (*p* < 0.05) in total heme pigment were found in the treated minced pork after 9 days of refrigeration storage compare with previous determinations (0, 3 and 6 days).

Heme pigment conversion degrees for all minced pork treatments are presented in Table 5. After 9 days of refrigeration, the results showed that the minced pork with PFJ (T1) produced an increase in the heme pigment conversion % equal to 32.00%. When HE was

added to PFJ, even the lowest HE concentration, 10 ppm GAE (T4) produced an increase in heme pigment conversion degree (47.99%), compared to the formulated sample with PFJ alone. So, compared to PC, the samples formulated with PFJ + HE provided increases in the heme pigment conversion degree in T2 and T3 or slower in T4 (Table 5). Moreover, PFJ + HE and SN + SA have the same effect on the heme pigment conversion degree, meaning an increased percentage. Results found for pH values, NO—heme and heme pigment conversion degree show important aspects related to sample formulation. The results can be attributed to the reducing and acidic properties of SA or HE that favour met(III)Mb reduction, NO generation, and the reaction of heme pigments with NO also.

#### *3.5. Residual Nitrite Level*

Nitrite added in the meat reacts especially with heme proteins (myoglobin, haemoglobin), and less with nonheme proteins, lipids, and carbohydrates. Unreacted nitrite, named residual nitrite, can be depleted by the formation of nitrogen gasses [46]. Nitrite depletion in cured meat is influenced by pH, temperature, time, meat type, salt, and nitrite concentration. The residual nitrite level in meat treated with PFJ and PFJ + HE is shown in Table 6. For all samples, residual nitrite level decreased during refrigeration storage, but the most rapidly decreasing was found after 3 days of storage. Throughout the refrigeration, the lowest residual nitrite level was found in the negative control (NC), without adding any nitrite, and the highest level was found in pork mince treated with PFJ alone (T1). The addition of HE to the PFJ favours the decrease in the residual nitrite level. Therefore, the residual nitrite level in minced pork treated with PFJ + HE decreased with increasing levels of HE. In the pork mince treated with the PFJ + 50 ppm HE (T2), the residual nitrite level was significantly (*p* < 0.05) lower than those found in the positive control. Regarding the residual nitrite level in the minced pork treated with the PFJ + 50 ppm HE (T3), the rate of nitrite depletion with storage time was similar to the one found for the positive control. The residual nitrite depletion was a bit slower in the meat treated with PFJ + HE, at level 10 ppm GAE (T4), compared with the PC. The addition of HE showed a statistically significant effect in the nitrite depletion with storage time, and so, the decrease in the residual nitrite level (*p* < 0.001).

**Table 6.** Changes in residual nitrite level and volatile basic nitrogen of minced pork depending on treatment and storage time.

#### **Table 6.** *Cont.*

a–e Means in a row without a common letter significantly differ. NC—negative control; PC—positive control; T1—treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4—treatment with PFJ +HE, 10 ppm GAE.

These results were in agreement with those found by other researchers [47,48], who reported continuous depletion of the residual nitrite level in cured meat during the storage time. A more pronounced decrease in residual nitrite level in minced pork formulated with SA and HE, compared to the sample formulated with PFJ alone may be the result of the pH decreasing and the interaction of nitrite with the SA and the HE. Some researchers reported a decrease in free nitrite in aqueous solutions of nitrite and sodium ascorbate [49]. Other results suggested that NO resulting in the reaction of free nitrite with SA is responsible for nitration reactions and residual nitrite depletion during meat curing [50]. A good correlation (*p* < 0.001) was found between pH value and residual nitrite depletion rate and between HE concentrations and residual nitrite depletion rate. Similar results were achieved in different research regarding cured meat products [51–54].

#### *3.6. Volatile Basic Nitrogen (VBN) Value*

Volatile basic nitrogen is a product of bacterial spoilage and endogenous enzyme action [55]. VBN is mainly composed of ammonia and primary, secondary, and tertiary amines [56] resulting from amino acid degradation [57,58]. The changes in the VBN value of all samples with storage time are shown in Table 6.

The results show the progressively increasing VBN for all samples. The highest VBN values were found in NC and the lowest in the PC, throughout the refrigeration period. In pork mince formulated with PFJ alone, VBN values were lower than those found in NC but higher than those found in PC. Meat formulation with PFJ + HE decreased VBN values in total phenolics in HE concentration-dependent manner, the lowest values were found for PFJ + HE, in the concentration of 50 ppm GAE (T2) and the highest for PFJ +HE, 10 ppm GAE (T4). In the T2 samples, the geometry of the VBN values concentration dependence was similar to one obtained for PC, but the values were higher by an average of 11.14%. A previous study used fermented spinach for pork loin curing and reported similar results [15]. VBN values in samples treated with fermented spinach were higher than those found in the negative control and lower than those found in the positive control treated with synthetic nitrite.

#### *3.7. Microbiological Analysis*

The effect of PFJ and PFJ + HE on total viable count (TVC) is shown in Table 7. The initial number of bacteria in samples was between 2.60 log CFU/g and 2.77 log CFU/g which indicated the good quality of pork used in this study. For all samples, TVC increased with storage time and the value of NC and T1 increased faster than PC, T2, T3, and T4. From day 0 to day 3, there were strong significant differences between NC and T1 (*p* < 0.05). After 6 days of storage, significant (*p* < 0.05) differences between NC and T1, which indicated that PFJ inhibited the growth of TVC alone, were found. After 6 days of storage significant differences (*p* < 0.05) between T1 and samples T2 were found and these results indicated that HE added in PJF increased the antimicrobial activity of PFJ. A significant correlation was established between PC and samples T2, T3, and T4 (*p* < 0.001).

**Table 7.** Changes in total viable count of minced pork depending on treatment and storage time.

a–e Means in a row without a common letter significantly differ. NC—negative control; PC—positive control; T1—treatment with PFJ; T2—treatment with PFJ + HE, 50 ppm GAE; T3—treatment with PFJ + HE, 25 ppm GAE; T4—treatment with PFJ +HE, 10 ppm GAE.

After 9 days of storage in all samples, TVC was higher than 5.69 logs CFU/g and so, not acceptable for safe consumption, but TVC values were lower for T1, T2, T3, and T4 than NC. Similar results were reported by the literature, showing that fermented spinach extract, celery juice powder, and cherry juice powder can inhibit the growth of microorganisms in different meat models [15,59]. Other researchers tested the antimicrobial activity of the ethanolic extracts obtained from rosemary and cloves and found important antimicrobial activity for extracts used alone or in combination [31].

#### *3.8. Correlations between Analysed Parameters*

The Pearson correlation coefficient (r) is the simplest way to measure a linear correlation. It takes values between −1 and 1 and indicates the relationship between two variables in strength and direction. When one variable changes, the other variable also changes. In this study, we analysed the effects of various concentrations of HE in combination with a constant PFJ concentration on the pH, fatty acids profile, TBARS value, NO-heme, total heme pigment, heme pigment conversion degree, residual nitrite, VBN, and TVC. pH value is a very important parameter for pork mince and was influenced by VBN value (r = 0.2488, *p* < 0.0001) and TVC, in a positive and linear manner. NO-heme is a parameter related to meat colour stability. According to the results, a weak positive relationship was found between NO-heme and TBARS (r = 0.9349, *p* < 0.0001), but negative correlations were found between TBARS value and MUFA (r = −0.7275, *p* < 0.0001), and between TBARS value and PUFA (r = −0.8643, *p* < 0.0001). Colour stability is influenced by TBARS and could be related to fatty acids profile. A negative correlation was found between NO-heme and residual nitrite (r = −0.7786, *p* < 0.0001). The pH values determined in the present study established positive correlations with heme conversion degree, (r = 0.5848, *p* < 0.0001), and NO-heme (r = 0.5787, *p* < 0.0001) (data in Table S1).

#### **4. Conclusions**

The use of PFJ in combination with HE exerted an inhibitory effect on the growth of spoilage bacteria that metabolize nitrogen compounds and significantly increased the stability of unsaturated fatty acids to oxidation in minced pork. The addition of HE in the minced pork decreased the TBARS values compared to T1. At any time during the experiment, NO-heme increased in a concentration-dependent manner with HE. So, HE may influence the NO-heme concentration. The concentration of HE of 25 ppm together with PFJ was the most effective to delay the FA oxidation in pork mince, whereas the higher concentration of HE (50 ppm GAE) had decreased the total PUFA and increased TBARS, suggesting a prooxidant effect on the minced pork lipids. Colour and lipids stability may depend on the HE and PFJ combination. However, T1, T2, T3, and T4 showed lower TVC values than NC. Between them, T2 was the most effective against microbial growth. In conclusion, these combinations of natural sources of nitrites and antioxidants could be considered promising meat alternatives to synthetic nitrites and antioxidants.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/agriculture13020432/s1, Table S1: Pearson's correlation coefficients of analysed parameters.

**Author Contributions:** Conceptualization, C.P. and C.N.P.; methodology, C.N.P. and B.T.; software, M.S. and G.S.; investigation, C.P. and C.N.P.; resources, C.P.; data curation, C.N.P.; writing—original draft preparation, C.P.; writing—review and editing, C.N.P.; visualization, M.S.; supervision, A.E.U.; project administration, C.P. and G.T.; funding acquisition, C.N.P. and C.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Romanian UEFISCDI projects PN-III-P2-2.1-PTE-2021-0508, grant number 71PTE/2022.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Review* **Transforming** *Rhodotorula* **sp. Biomass to Active Biologic Compounds for Poultry Nutrition**

**Daniela-Mihaela Grigore 1,2, Mădălina Ungureanu-Iuga 3,4,\*, Elena Narcisa Pogurschi 1,\* and Narcisa Elena Băbeanu 2**

**Abstract:** In broiler chick-rearing, the color is usually acquired by synthetic carotenoids in addition to broiler diets (25–80 mg/kg feed), often represented by β-apo-8 -carotenal. In the past fifteen years, the demand for organic food products originating from free-range reared chicks started to grow, with a more directed awareness of the quality of meat and egg. Various investigations have been reporting microorganisms, such as the oleaginous red yeasts genus *Rhodotorula* sp., as fast-growing unicellular eukaryotes able to synthesize natural pigments. *Rhodotorula* sp. represents a perfect choice as a natural resource due to the capacity to adapt easily to the environment valuing low-cost sources of nutrients for their metabolism and growth. The biodiversity and the ecology effects establish novel boundaries regarding *Rhodotorula* sp. productivity enhancement and control of biological risks. It is, therefore, necessary to review the current knowledge on the carotenoid synthesis of *Rhodotorula* sp. In this paper, we aimed to address the pathways of obtaining valuable yeast carotenoids in different conditions, discussing yeast biosynthesis, bioengineering fermentative evaluation, carotenoid extraction, and the techno-economic implication of valuable pigment additives on poultry nutrition. Finally, the pro-existent gaps in research are highlighted, which may clear the air on future studies for bio-carotenoid engineering.

**Keywords:** artificial pigment alternative; broiler nutrition; carotenoids; health; pigment additives; vegetal waste

#### **1. Introduction**

Carotenoids are soluble pigments classified as tetraterpenoids divided as primary (hydrocarbons, carotene) and secondary as their oxidation product (xanthophylls). Widely, around 1100 different carotenoids [1] are synthesized in plant, algae, and fungi species. As natural lipophilic pigments [2], they are often characterized by a range of colors, starting from a pale and creamy yellow, light-pink, strong yellow, pink, and orange until strong red pigmentation and a rare, purple color [3]. Under natural circumstances, carotenoids have a multitude of roles, including sustaining photosynthesis, ensuring photoprotection [4,5], antioxidant capacity [6], reproductive enhancement [7], embryonal development [8], cell maturation [9], and immune system protection [10]. Birds cannot synthesize carotenoids hereby; carotenoids must be included in dietary intake. Dietary feed ingredients used in

**Citation:** Grigore, D.-M.; Ungureanu-Iuga, M.; Pogurschi, E.N.; B ˘abeanu, N.E. Transforming *Rhodotorula* sp. Biomass to Active Biologic Compounds for Poultry Nutrition. *Agriculture* **2023**, *13*, 1159. https://doi.org/10.3390/ agriculture13061159

Academic Editors: Vincenzo Tufarelli and Lin Zhang

Received: 24 April 2023 Revised: 26 May 2023 Accepted: 28 May 2023 Published: 30 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

commercial poultry feeding formulas are often processed in pelleted and extruded form, with consideration to nutrient availability and economic efficiency [11]. Mechanical procedures such as palletization or extrudate are currently employing high-temperature and pressure applied directly to feeding ingredients [12], thus affecting the retinol and retinol precursor by degradation, cumulating the vitaminic losses through handling and storage [13].

Carotenoids represent significant resources of retinol precursors (0.66 μg β-carotene=1U of retinol acid), with large implications for healthfulness and quality [14] products. Poultry is the most successful livestock sector around the globe and tends to grow due to the increasing consumption of poultry products. By the year 2020, the poultry sector generated almost 101 metric tons of meat and 1.65 billion eggs [15]. The great potential of carotenoid sources in industries (including food, feeds, nutritional supplements, pharmaceutics, and cosmetics) will have increased the forecasted market value to around \$2.0 billion by 2022 [16]. The most commonly used food and feed colorant additives in poultry nutrition are xanthophylls (lycopene, canthaxanthin, astaxanthin, and zeaxanthin) that originate from almost 90% mainly synthetic resources. Annually, the market for pigment additives tends to grow by 8.2% percent during the forecasted period 2022–2032 [17] due to the increasing consumption of poultry products (meat and eggs). Currently, there has been a growing interest in obtaining organic pigment additives from non-conventional resources (algae, bacteria, and yeasts). The composition and the stability of the natural resources might be undefined and wide because of the complexity of biochemical metabolism and biological variability that is often associated with the cell structure. Great consideration was attributed to the carotenoid biosynthetic pathways of yeast, understanding the carotenoid yield, as productivity and integrity, with a view regarding product improvement and industrial scalability. In non-phototrophic microorganisms, carotenoids present a clear advantage in obtaining natural pigments [18]. One of the most important attributes is the capacity of microorganisms to use industrial waste as raw material substrate [19], hence increasing profitability and lowering the related costs of production. Many microorganisms synthesize carotenoids and present a valuable industrial potential (Table 1), although the data concerning *Rhodotorula* sp. yeast pigment application on livestock nutrition are few.

**Table 1.** Carotenoid-producing microorganisms.

**Table 1.** *Cont.*

The current paper aims to highlight the multitude of approaches to obtaining valuable yeast carotenoids in different conditions, discussing yeast biosynthesis, bioengineering, fermentative evaluation, carotenoid extraction, and the techno-economic implication of valuable pigment additives on poultry nutrition.

#### **2.** *Rhodotorula* **sp. General Aspects**

The genus *Rhodotorula* sp. covers more than 165 species [34]. Morphologically, *Rhodotorula sp*. is a polyphyletic-shaped yeast [35] forming fast-growing colored colonies [36]. The proliferation of the *Rhodotorula* genus is generally regarded as asexual [37]; however, some strains belonging to the genus present sexual reproductive traits [38]. *Rhodotorula sp*. ecology and biodiversity cover a board of environmental varieties using a large variety of carbon resources, including glycerol [39], glucose [40], sucrose [41], galactose [42], and maltose [43], often encountered as dominant in yeast microflora (water, soil, vegetal, and animals) [44].

Yeast such as *Rhodotorula* sp. represents a perfect choice as a natural resource of secondary metabolites (Figure 1): carotenoids [45], lipids [46], and extracellular enzymes (Table 2). Saprophytic and ubiquitously found, the *Rhodotorula* genus possesses a full capacity for intracellular carotenoid biosynthesis [47] (provitamin A precursors, such as β-carotene and γ-carotenoid) [47,48], although the main carotenoids are torulene and torularhodin [49].

**Figure 1.** Major benefits of bioactive compounds from *Rhodotorula* sp.

Carotenoids are mainly synthesized via successive condensation (Figure 1) attributed to isoprenoid units such as isopentenic pyrophosphate (IPP) isomerized in dimethylallyl pyrophosphate (DMAPP) [4,58–63]. Particularly, yeasts such as *Rhodotorula* sp. possess the ability to transform lycopene into cyclic carotenoids like β-carotene (under lycopene β-cyclase action) and γ-carotene (conversion supported by lycopene cyclase).

The γ-carotene unit represents the main precursor for yeasts' carotenoid formations, as shown in Figure 2. β-carotene (C40H56) is the most common and abundant

precursor for retinol [64], strong-orange-red colored, chemically classified as isoprenoid (synthesized from eight isoprenoid units) [65]. Torularhodin is regarded as a xanthophyll (C40H52O2—3 ,4 didehydro β, ψ-carotene-16 oic acid) due to the presence of the carboxyl group [66] and represents the prevailing chemical structure in *Rhodotorula* sp. total carotenoid yield [67]. Torulene is classified as a carotenoid. The torulene molecule includes only hydrogen and carbon atoms [49], C40H54, 3 ,4 dihydro-β, ψ-carotene.

**Figure 2.** Rhodotorula carotenoid synthesis via MVA pathway; adapted after [68].

Concerning pigment bio-applications, *Rhodotorula* sp. yeast owns valorous advantages: fast-growing capacity, usually on organic waste materials (carbon-rich materials), and costeffective production and harvesting (minimized human/process-associated interventions), which is more than suitable for large-scaling pigment industrial production and directly competing within the pigment market.

The vegetal sector is the most important resource concerning natural pigment industries. Factors such as poor soils [69] and climate-changing conditions [70] are currently affecting the industries, leading to long delays and negative economic implications within the pigment-related industries (food, feed, pharmaceutical, and cosmetic) [71]. Furthermore, the legislation and regulation of synthetic pigment use in food and feedstuff are narrowing the offer. Microbial pigment additives are still at the developing stage, up-scaling as the future organic source, an alternative to conventional resources, proving higher pigment capacity, in a shorter time. Recent studies regarding pigment-producing yeasts such as *Rhodotorula* sp. show an improvement in carotenoid productivity and componence that might be modulated [72] by mutagenesis [73] combined with other techniques, including different controlled stressors, such as temperature [74,75], substrate composition [76], lightning conditions [77] and aeration [78]. β-carotene, torularhodin, and torulene are valorous compounds synthesized exclusively by fungi and yeast [79], found in lipid bodies

of cellular biomass, and they are light unstable. The yeast carotenoid compounds present highly anti-oxidative characteristics [80].

#### **3. Factors Affecting Pigmentation**

#### *3.1. Yeasts Nutrition*

The productivity efficiency is generally based on the interdependency of the specific pathway involved and the culture medium (organic or inorganic compounds), strain specificity, and the growing conditions [81]. It is essential to balance the yeast requirements and cultivation conditions, to support the carbon-efficient use and yeast growth rates, thus optimizing secondary metabolites yields [46]. Moreover, individual metabolites yield (pigment quality and quantity) depends on the enzymatic complex employed in the yeast metabolic system [82]. Carbon and nitrogen have been considered the main sources of energy [83], and growth support of microorganisms, metabolite enhancement, and carotenoid producibility could be modulated by balancing the substrate composition, although, there are studies that show the induced stress by nutritive limitation (nitrogen sources) enhances carotenoid productivity (up to 0.75 mg/g dry cell weight) in Rhodotorula toruloides by suppressing the cell growth [84].

The influence of nutritional carbon/nitrogen resources on yeast fermentation and carotenoid yield might differ (Table 3) under the strain pigmentation capacity. Moreover, the nutritional carbon source might modulate the yeast carotenoid profile. Li et al. [83] showed that a medium containing glucose had more than 93% torularhodin in the total yeast carotenoid profile. There are also differences between the utilization of organic and inorganic compounds. Organic nitrogen resources such as yeast extract improve carotenoid yield up to 987 g/L [83], although using residual waste such as fruit and vegetable pulp/peel [85] and beer sludge represent hardly recycled market resources in developing countries, presenting worthy yeast nutritional potential.

**Table 3.** Factors affecting the carotenoid metabolism in yeast.

160

#### **Table 3.** *Cont.*

**Table 3.** *Cont.*

Sharma and Ghoshal [79] used onion peel, mung bean, and pea (agro-industrial wastes) as a substrate for pigment production on *Rhodotorula mucilaginosa*, obtaining the best carotenoid productivity (27.4 mg/L) on onion peel extract. The olive oil industrial waste (20%-culture media) improved the total volumetric carotenoid production (up to 5.5 g/L) [67]. Carrot peels or starch in the potato feed industry is a typical example of recoverable fractions either as solids or as sludge which, after drying and sterilization, can be included directly in yeast bioprocess as sources of carbohydrates [95]. By recycling vegetal waste and thus improving the culture media for yeast growth, the productiverelated costs are reduced to a minimum or absent in the development of the market economy. The costs involved mainly in recovering profitable nutrients from food waste processing; the credit played is derived from nutritional applications useful in all agriculture branches. At the same time, the economy of waste conversion and valuable byproducts generates a new secondary-industry domain, with new jobs and skills at the place of production [96].

#### *3.2. Yeasts Fermentation Conditions*

Carotenoid yield related to obtaining secondary specific metabolites might be an induced response generated by different stressors applied to the *Rhodotorula* sp. growth (nutritive limitation, aeration, and temperature) and could influence (by delaying or accelerating) the carotenoid synthesis. It has been demonstrated that the yeast carotenoid yields maximum values within reaching the cell's mature development [97]. Furthermore, the variability of the carotenoid yield componence proportions within the mature cells variates depending on the temperature and time of cultivation, 144 h on yeast malt, 252.99 μg/g total carotenoids [98], 120 h on yeast malt, 223.5 μg/g total carotenoids production [99].

The temperature parameter is a critical cultivation factor in the first place, affecting culture viability and biomass productivity and active bio compounds quality. Temperature correlates with metabolic functions and influences enzymatic activity [100] with carotenoid productivity, hence, effective regulation between cyclic carotenoids synthesis, followed by precursors. An indirect metabolic synthesis between the low temperature of β-carotene synthesis and the opposite [101], increasing xanthophyll's and β-carotene precursors concentrations at higher temperature values, is probably by the low-temperature enzymatic activity of lycopene β-cyclase. Recent research points out that higher values of β-carotene production were recorded-by at 20 ◦C 250 mg/L representing 92% of total carotenoids compared with 30 ◦C, 125 mg/L, and the amount of 60% from total carotenoids and 35 ◦C, with less than 19% β-carotene and torulene encountered in biomass; although at 35 ◦C the torularhodin synthesis increased, leading up to 78% of total carotenoids in biomass [29,39,102].

Yeasts such as *Rhodotorula* sp. have naturally developed a light-sensitive response to environmental lightning conditions, protecting the yeast cells by synthesizing a large amount of β-carotene. White light irradiating trials were conducted on 21 strains of *Rhodotorula* sp., and the results concluded that the amount of carotene is twofold higher by irradiation (14.2 mg/100 g dry weight biomass). At the same time, light irradiation as a photo-regulative measure could modulate yeast growth and biochemical componence to enhance carotenoid productivity [77], although strong light exposure could negatively affect the yeast cultures, inhibiting their growth.

*Rhodotorula* sp. is an oxygen-dependent yeast [103] affecting both viability and productivity. Recent studies regarding the oxygen demand have demonstrated that the yeast cell growth and metabolism are strongly crisscrossed with yeast phenotype and the yeastapplied stressors, confirmed by the secondary metabolite's yields [30] and other bioactive compounds such as hemoproteins [104].

Besides the photo-protective role, yeast carotenoid active compound has an oxidative protection function facing the oxidant agents before yeast cell wall attack [105]. Oxygen supply, through aeration, agitation, or airlift bioreactors, is crucial to yeast metabolite productivity. Yeast oxygen requirements concerning carotenoid productivity were studied, and the results show an increase of end-metabolites synthase (torularhodin) expected from cyclic carotenoid oxidation [106].

#### **4. Yeasts Pigment Extraction and Quantification**

Yeast carotenoid yield determinism is directly modulated by the yeast phenotype and the engineering approach via metabolites enhancement. Despite the progress achieved in the biotechnological yeast carotenoids synthesis optimization, there is a permanent need for research efforts to constantly adapt and improve the in-process efficiency and minimize the economic implication. The yeast fermentative process is followed for the quantification of productivity determinations: preparative (harvesting, cell biomass disintegration) and quantitation methods (extraction, separation, and evaluation).

Harvesting viable cells, carotenoid extraction and purification of the carotenoid components are the most expensive procedures in techno-economic analysis. There are many ways to process yeast carotenoids. Harvesting cell biomass can be easily achieved by mechanical, chemical, or biological strategies. The centrifuge separation is the conventional mechanical method used in yeast industries, employed at 8000–10,000 rpm during a period of 7–10 min [107–109]. Current innovative methods concerning biomass harvesting are flocculation, pre-concentration techniques, high-pressure filtration, flotation, osmosis, bubble columns, and exploitation of hydrophobicity/hydrophobicity yeast proprieties [110]. The appropriate harvesting method is generally chosen through yeast proprieties such as cell size, biomass density, production volume, and final product specificity. Consecutive in yeast recovery, the yeast cell purification techniques, as successive washing with solvent and filtration cycles with the purpose of cell biomass clear separation. There are many carotenoids extractive methods [30,111–113] for samples and pure specific carotenoid quantification. Microbial carotenoids are secondary metabolites [114], present in almost 95% of the cell. Cell wall disruption and disintegration are needed as preparative procedures in carotenoid extraction. The most common approach is an organic/solvent-free mechanical breakage [115,116] combination between sonication/pressure treatments or

freeze-thawing/sonication [117,118] without having major losses on the yeast cell biomass compared to the synthetic chemical disruption that might generate artifacts or radicals [119], artificial condensation (acetonides) [120] or at worst, generating radioactive components (aldehydes) [121]. There are cell breakage methods that are less harmful, involving hydrolysis, supercritical CO2 [122], or enzymatic digestion extraction [123,124], having superior recovery rates, and implying extra financial costs. Carotenoids are non-polar chemical compounds characterized by water insolubility. A more hydrophilic carotenoid form is represented by their derivates, xanthophylls, due to the hydroxyl radical on the chemical structure. The commonly used extractive processes imply the reagents (acetone, cyclohexane, dimethyl sulfoxide, chloroform, petroleum ether, and ethyl acetate) usage as extractive solvents to separate the pigment compound in the partitioned liquid of analysis [125,126]. Carotenoids and xanthophylls are chemical compounds having more than nine double bonds that are capable of light absorption, detected between visible/UV wavelengths range of violet and blue-green spectra (450–550 nm) [127], naturally reflecting red, orange, and yellow color shades. Carotenoid detection and quantification have various protocol approaches, employing spectrophotometry or spectroscopy determinations. Pigment quantification assays are practically based on a comparative determination against pure chemically carotenoid materials (commercially available standard references, as 95–99% pure, for specific determinations), lab standardized as etalons curves, as for accuracy, reproducibility, and repeatability (as for peaks, retention time and area of peak) that later on might be interpreted as values using conversion formulae [128]. Carotenoid UV-Vis assay is a feasible method of quantification but needs a long time to determine because carotenoids obey the law of Lamber–Beer (the compound concentration is directly proportioned with the compound spectral absorbance) [129]. The UV-Vis conducted assays evaluate the liquid carotenoid sample (up to 3.0 mL) compound against the pure carotenoid standard reference substance with the intention of total carotenoid measurement [130]. The disadvantage of employing the UV-Vis method is the mediocre specificity consisting of the incapacity of distinction between individual carotenoids (similarity of peaks and absorbance wavelength around 459–500 nm for more than four distinct carotenoids). Quantitation is possible only by mathematical determination by using specific carotenoid partition coefficients [131]. A more precise approach is using the HPLC method (high-pressure liquid chromatography). Despite the time and costs regarding reagents and capillary system components, carotenoids are detected and measured simultaneously and accurately quantified individually [132] needing no more than 1.5 mL of liquid sample, injected (40 μL) with high pressure, carried (flow rate: 0.5 mL/min) with the eluent (A: acetonitrile: water, 9:1 and B: 1% formic acid ethyl acetate) to the stationary component (column C18, 250 mm × 4.6 mm, 5 μm) and detected (UV detector) [88]. Moreover, it highlights the labor exercise that lies in the systematic examination of the spectral signature, which is no longer just that of the compound of interest, needing specific determination to identify and quantify impurity componence. The FTIR (Fourier transform infrared spectroscopy) is capable of simplifying the total carotenoid quantitation, not only by time (less than 150 s) and cost but also by accuracy, dividing them as chemical structures [133]. RAMAN spectroscopy is the superior method of determination, analyzing at the same time light absorbance and matter structure of the sample only by photon laser interaction with the small sample size, in a very short time determination—based on the relation of light interaction on all materials, scattering the same amount of energy as incidence light [134].

#### **5. Yeast Carotenoids in Poultry Nutrition**

#### *5.1. Retinol Requirements and Retinol Precursors in Poultry*

The challenge regarding poultry vitamin requirements is and will be an actual research domain due to the genetic abundance and oscillational nutritional aspects between various factors that appear in poultry-intensive sectors (health status, veterinary medications, feed, breed, age, housing aspects, and rearing technology). Poultry specialized hybrids have exigent vitamin A requirements (Table 4), solidly correlated with the breed's purpose and

rearing management recommendation. In poultry, both layers and meat broilers have an excessive level of vitamin A feed supplementation starting from 10,000 International Units (IU)/kg diet up to 13,000 IU/kg diet, according to supplier recommendation, despite the requirements profile established by the National Research Council (NRC, 1994) colorant additives should not exceed 4500 IU/kg-fed meat broilers and 2500 IU/kg-fed layers. Moreover, vitamin supplementation is recommended to be equal to or more than birds' requirements [135], hence avoiding vitamin deficiency. In poultry, provitamin A and retinol deficiency could be a consequence of malabsorption or the impossibility of metabolic conversion, often regarded as biologically available [136]. The effects concerning retinol deficiencies are complex and affect a large range of metabolic activities: weight loss cumulated with slowing down the growth processes and negative performance rates [137], follicular hyperkeratosis, epithelial lesions, xerophthalmia [138], keratomalacia, hemeralopia, reproductive system malfunction [139], and gastrointestinal disorders. Exceeding retinol and provitamin A in poultry leads to xanthomatotic disorders [140] and hypercalcemia, followed by bone system disorders. The vitamin A origin and the stability within the dietary intake is a current challenge, although most of the commercial feeding formulas are developed and balanced by adding artificial vitamins along with micro and macro elements and by not taking into account the vegetal raw material vitamin content, thus the vitamin antagonistic [141] or destructive compounds [142]. Additionally, naturally occurring vitamins in feed and forages are presenting stability issues [61] due to inadequate feed manipulation and storage, often causing vitamin oxidation and frequent bacterial infestation [143], implying constant economic depreciation and loss [144]. Furthermore, the treatments such as insecticides and pesticides administrated to livestock crops are interfering with and affecting the feed vitamin concentration, leading to toxic traces within grain cultures [145].

**Table 4.** Broiler chicks and laying hens' pigment additive (IU/kg fed) in dietary-fed formulas \*.

\* As supplier nutritional guidelines.

#### *5.2. Carotenoid Absorption in Poultry*

The physiologic and biochemical roles of provitamin A and retinol precursor cover multiple functions. In poultry, in the starter growing phase, retinal and provitamin A stimulates the growth processes and normal development of the reproductive system [158]. Provitamin A is the most important in preventing epithelial disorders (conferring elasticity

and anti-infectious resistance) and maintaining homeostasis of the visual function [159]. In poultry physiology, carotenoid synthesis is absent. Therefore, an exogenous intake is required. Birds can synthesize retinol from β-carotene through the retinal enzyme [90,160], β, β-carotene-15,15 -monooxygenase, capable of separation into two retinal symmetrically molecules [161]. The vitamin A precursor, β-carotene is an indispensable nutrient for reproduction, growth, and production (the biological activity is almost 60% of retinol activity). β-carotene absorption and bio-disposable variates by the bird's metabolism, the bird's absorptive capacity, and the forage quality related to formula stability and biochemical characteristics. Physiologically (Figure 3), β-carotene is a long-term absorption compound (up to three days until retinol conversion) that combines into chylomicrons in the small intestine mucosa (duodenum) and is carried further to the liver through the portal vein [162]. Oil presence enhances the vitamin A precursors absorption and liver metabolization [163], combined with lipoproteins in triglycerides (VLDL and LDL) and transferred to a specific tissue (skin, meat, fat, ovary, and egg yolk). Retinol in excessive quantity is moreover deposited in the liver and blood, then in muscle, fat, eggs, or skin [164]. Egg yolks' carotene deposits vary between 40–50% of total carotenoid intake [165]. However, most of them are lycopene, canthaxanthin, astaxanthin, and zeaxanthin, and lidding the β-carotene yolk concentration less than 1% due to the higher xanthophyll absorption in the bird's digestive tract [166]. Moreover, the bioavailability of carotenoids is mostly influenced by the matricidal food structure, carotenoid compound chemical structure, and interaction with other dietary nutrients.

**Figure 3.** Carotenoids metabolism in poultry physiology; adapted after [167].

#### *5.3. Poultry Feed Sensorial Additives*

Nine pigment additives (Table 5) are regulated and used widely in the EU (indexed as pigment additives E160, E161, E162, and E163) [168] as appropriate for poultry nutrition labeled (EU Council directive 96/23/EC, 2010) as dietary pigment additives (SafeFood, 2022), for improving the egg yolk skin and meat product color.

**Table 5.** Pigment additives used in poultry nutrition 1.

1 Pigment additives regulation in poultry feed approved by the E.U.; 2 additives encoding by Council directive 70/524/EEC; \* expressed as mg/kg diet.

The use of commercial carotenoids in poultry feed formulations is expensive and originates from around 90% artificial sources. Most of the pigment additives are approved for dietary inclusion up to 80 mg/kg in broiler chicks feeding formula [11], except canthaxanthin. Canthaxanthin levels are restricted and should not exceed more than 8 mg/kg for laying hens and 25 mg/kg feed for broiler chicks [169]. As the sole pigment additive used in human, fish, and poultry nutrition, canthaxanthin [170] dietary overdosage leads to residual pigment tissue deposits that expose the final consumer to a pigment intake that exceeds the Acceptable Daily Intake (ADI, 0.03 mg/kg body) [171], and might negatively affect the consumer's health (high risk of toxicity). Research concerning natural carotenoid sources as an alternative to commonly used synthetics for livestock nutrition shows that using natural resources such as maize and pasture (fresh or preserved) [172,173] and genetically modified organism (GMO) or non-GMO (plants, algae, and yeasts) could serve as superior native carotenes used pigment additives [174–176]. Few studies regarding the microbial piments additives on broiler meat [45,177,178]. Dietary inclusion of red yeast *Phaffia rhodozyma* (10–20 mg/kg feed) on broiler chicks positively affected the broiler chicks' performances and immune response, presenting 10 times stronger pigment capacity [179]. Moreover, in broiler nutrition, pigment additives are often employed along with oils [180,181] to mitigate the spontaneous oxidative effects on fat deposits and to improve the carcass's oxidative stability [182]. Furthermore, dietary carotene addition shows controversial effects via vitaminic metabolism, showing antagonistic [183] and synergic action [175] and might have an opposite role as an antioxidant [184] and pro-oxidant factor [185], depending on factors such as dietary formulation (inclusion or addition). The antagonism between vitamin E accumulation and β-carotene was studied, and the results show that the presence of β-carotene in broiler breast meat tends to limit vitamin E accumulation [186]. However, the dietary addition of lycopene and vitamin E improves the broiler chicks' growth performance and tight meat oxidative stability and also presents a synergic benefic effect on thigh meat cholesterol content. In laying hens, diets include distinct amounts of corn and alfalfa meal, contributing to the content of native pigments in the diet [6]. Intensive rearing systems diets are low in native xanthophylls. Therefore, the egg yolk is often characterized by a pale-yellow color [6] due to rich amounts in barley, rice, or wheat that are supplemented with artificial pigments (β-apo-8ícarotenoic-acid-ethyl ester) to satisfy the range of color scores required by the European egg producers and to meet the consumer's expectations [187].

#### **6. Conclusions**

As a directed movement in the food and feed markets guided for more natural products, the demand for organic ingredients is rising. Feed formulation recipes using natural and organic additives are the new trend in livestock nutrition research, using not only active principles that affect vegetal but also microorganisms for valuable active bio compounds. Yeast pigments are outstanding sources of natural color, covering a wide range of nutritional and medicinal properties. Both carotenoid yield and total carotenoid structure are important aspects that could be optimized depending on strategy, adopting strain genetic engineering and process development, and employing cheap organic substrates. Further studies are required to establish biological and chemical proprieties, and yeast carotenoid mechanisms, enhancing yeast carotenoid productivity, stability, and marketability as alternatives to classic synthetic pigments. Data generation concerning a highly productive yeast process involving scalability for large-scale adaptability to fermentation aspects (fermentation design and bioreactor types) is essential. Furthermore, studies regarding the effects of value-added yeast pigment additives on livestock health, productivity, and product quality are important in validating nutritional and medicinal potential. Not last, consumers' perceptions and preferences in buying animal products obtained with microbial pigment additives firmly increase the need for knowledge.

**Author Contributions:** Conceptualization, D.-M.G. and N.E.B.; methodology, M.U.-I.; validation, E.N.P. and N.E.B.; resources D.-M.G.; data curation, D.-M.G., and N.E.B.; writing—original draft preparation, D.-M.G. and N.E.B.; writing—review and editing, M.U.-I.; visualization, E.N.P.; supervision, N.E.B.; funding acquisition, E.N.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** The article processing charge was supported by the University of Agronomic Sciences and Veterinary Medicine of Bucharest, the Faculty of Animal Productions Engineering and Management.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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**5. Insect growth regulators**

#### **5.1. Juvenile hormone analogs (methoprene and pyriproxyfen)**

Methoprene, hydroprene, kinoprene, and triprene were synthesized in 1960s. These insect growth regulators interrupt juvenile hormone balance during the transition from the late 4th instar larvae to pupae and adults. Most mortality occurs at pupal stage or incompletely emerged adults. Another juvenile hormone analog pyriproxyfen was synthesized in 1970s, the IRG activity of which is much higher than methoprene [80]. The earliest experimental studies on potential of resistance development in mosquitoes to juvenile hormone analogs were in 1973 [82]. The collective results indicated low risk of resistance development [82–86]. For example, the selection of *Cx. quinquefasciatus* by methoprene for 10 generations only lead 3.9 to 21.3-fold of resistance [86], while the selection of *Cx. pipiens* for 8 generations only resulted in 8- to 13-fold resistance to methoprene and cross-resistance to triprene [83]. Higher levels of resistance to methoprene did not necessarily occur in response to longer period of selection. *Culex tarsalis* Coquillett developed 86-fold resistance after 62 generations of selection [84], while 218-fold of resistance was achieved in *Cx. pipiens* after 40 generations of selection. In the latter case, selected mosquitoes were also cross-resistant to hydroprene and triprene, but not to diflubenzuron [85]. Rapid discharge and reduced detention of methoprene in mosquito tissue played an important role during entire process of resistance development, while metabolic detoxification seemed related to development and maintenance of high level resistance [87, 88].

Data are meager with regard to resistance development in wild populations of mosquitoes. *Aedes taeniorhynchus* (Wiedemann) in Florida, USA, showed 15-fold resistance after applica‐ tions of methoprene product during 1989 to 1994 [89]. Methoprene tolerance in *Aedes nigro‐ maculis* (Ludlow) was discovered in central California, USA, after 20 years of treatment. Control failure was encountered during 1998–1999 [90], where resistance levels reached thousands of fold [91]. The documented resistance seemed not related to the metabolic detoxification by P450 monooxygenase and carboxylesterase, and treatments using *B.t.i*. partially restored the susceptibility to methoprene [91]. Another reports in wild populations showed that 4.7- to 16-fold in *Cx. pipiens* in Cypress [15] and 9- to 54-fold in *Cx. quinquefascia‐ tus* in southern California [81]. Limited data showed very low risk of resistance to pyriproxyfen in mosquitoes [92].

#### **5.2. Chitin synthesis inhibitor (diflubenzuron)**

Diflubenzuron was synthesized in mid 1970s by Philips-Duphar B.V. This compound is a nonselective chitin synthesis inhibitor that interrupts formation of exoskeleton, interferes with integrity of cuticle, and causes leakage of body fluid and ultimately mortality of target organisms. Diflubenzuron acts on all stages of the mosquito life cycle, larval stages in partic‐ ular, younger larvae show higher susceptibility. Up to date, studies on resistance management are limited to laboratory populations. For instance, *Cx. pipiens* developed 7-fold resistance to diflubenzuron in response to selection for 5 generations [85]. *Culex quinquefasciatus* collected from the east coastal area in Dar es Salaam, Tanzania, developed 2.4- to 6.6-fold resistance after 10 generations of selection [86]. *Aedes aegypti* developed 3.3-fold resistance after 10 generations of selection, of which the resistance level increased to 8- to 20-fold after this population was hybridized with a mixing collection from 35 locations and then selected for 5 generations [93]. In general, the risk of resistance development to diflubenzuron in mosquitoes is relatively low.


*2.7. Expression Profiling of the M. sativa HSF Gene in Different Tissues*

To determine the expression patterns of individual *MsHSF* genes in various tissues, a hierarchical clustering heat map was also constructed from public RNA-seq data obtained from NCBI for exploring the transcriptional patterns of *MsHSF* genes in this study (Figure 9). The blue color in the graph indicates low transcript abundance and the red color indicates high transcript abundance. The analysis showed that there were different *HSF* genes showing a trend of high expression in six tissues of *M. sativa*. *MsHSF06* and *MsHSF15* were highly expressed in the roots. The expression of each gene was not significant in the root nodules. In pre-elongating stems, *MsHSF05*, *MsHSF09* and *MsHSF14* showed higher expression. However, the gene with higher expression in elongating stems was *MsHSF03*. In *M. sativa* leaves, *MsHSF07*, *MsHSF08* and *MsHSF16* showed higher expression. Interestingly, *MsHSF10* was not expressed in any other tissue parts of alfalfa but was expressed in the flowers, a phenomenon that suggests that different *MsHSF* genes have more obvious specificity in different tissues.

**Figure 8.** A schematic representation of the cis-acting elements identified in the 2000 bp promoter region upstream of the *MsHSF* gene. The different colors represent the number of cis-acting elements contained (**A**). The number of *MsHSF* genes corresponding to the cis-acting element (**B**).