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Effects of Microbial Transglutaminase on Technological, Rheological, and Microstructural Indicators of Minced Meat with the Addition of Plant Raw Materials

The aim of the study was to analyse the effects of transglutaminase on the physicochemical, technological, rheological, and microstructural indicators of minced meat with the addition of plant raw materials. The formulations of minced meat from beef trimming, hemp protein, and flax flour were optimized in terms of biological value and optimal content of essential amino acids. The addition of plant components in amounts greater than 18% caused an increase in the content of protein, fat, and ash in the minced samples. The rheological properties of minced meat samples without enzyme treatment changed depending on the proportion of plant raw materials. When the content of the flax flour was increased, the minimum ultimate shear stress and viscosity were observed, while the maximum values for these indicators were achieved in samples containing about 15% hemp protein, as well as in samples without plant additives. When adding transglutaminase to the formulation, increases in the ultimate shear stress and viscosity were proven for all combined minced samples. The combined minces, containing flax flour, had a more plastic structure and the lowest modulus of elasticity, while minces including 14% hemp protein or more than 87% meat components were identical to the control samples in terms of deformation and elasticity. In enzyme-treated minces, the plasticity of the samples reduced while density and elasticity increased. Transglutaminase treatment contributed to the formation of optimal technological properties of combined minces. Microstructural analysis showed the intermolecular bonds between protein particles in combined minced samples with the addition of enzymes. The research results have demonstrated the effectiveness of using transglutaminase in the composition of combined minced meat for the formation of a homogeneous and dense system with the necessary technological and rheological properties.

Introduction
One of the important directions by which to ensure public health is to expand the range of products for a healthy diet, which are characterized by high nutritional and biological value and include essential components in balanced ratios. A balanced diet provides full vitality and helps to prevent pathological disorders and human diseases. It is important to study approaches that use the application of plant raw materials with proven functional properties in the technology of combined products, which allows one to modify the composition and adjust the technological properties of food accordingly.
Due to the current deficit of animal protein in the world, the population's needs are being met using other sources, mainly of plant origin [1].
The increased requirement for protein sources and, at the same time, the need to ensure a rational diet have led to the emergence and development of a new direction in food production, namely, one of obtaining combined foods based on the potential resources of dietary proteins of both animal and plant origins. The combination of meat-and vegetable-based raw materials provides high nutritional value of processed products, increases the variability of formulations, leads to a homogeneous distribution of ingredients, minimizes losses during heat treatment, and ultimately contributes to the creation of products of stable quality.
The introduction of plant ingredients into minced meat is one of the ways in which one can obtain high-quality meat products with adjustable compositions and properties. However, it has mainly been proposed to introduce plant protein sources such as soy, chickpea, wheat, lentils, and peas into meat systems [2][3][4][5], even though, from the point of view of food chemistry, promising sources of protein include such ingredients as flax and hemp seeds.
Flaxseed is an important source of plant protein, which comprises 18 to 22 g/100 g of seed weight [6]. Flaxseed protein is composed of a salt-soluble and a water-soluble protein fraction. Flaxseed protein poses potential health benefits to populations due to high aspartic acid, glutamic acid, leucine, and arginine content and also its balanced amino acid profile [7]. Flaxseed proteins have a higher capacity to absorb water and oil than soybean protein isolates [8].
Hemp seeds have begun to be used in various foods with high nutritional value. Hemp protein showed a high degree of digestibility and hypoallergenic properties [9]. Hemp seed contains ω-3 and ω-6 essential fatty acids in optimal ratio and proteins (25 g/100 g dry weight) in which the amino acid profile includes all the essential amino acids [9,10].
In the traditional technology of minced products, the problem of obtaining a uniform product with homogeneous structure is solved by adding food additives, generally of inorganic origin (for example, phosphates). In the manufacture of combined products containing a significant amount of plant components, the phosphate content might be increased to achieve the necessary texture, which is unacceptable according to sanitary and hygienic standards, especially in healthy food product technology.
One of the approaches to solving the problem may be application the enzyme preparations to obtain a dense, homogeneous structure of the combined product. Transglutaminase (TG) produces inter-and intramolecular crosslinking bonds in the proteins [11]. Lantto et al. [12] noted works in which studies of the effects of TG on proteins of various origins are presented, e.g., about catalysing bonds between meat and soy proteins or between meat, casein, and gluten. Cross-linking proteins containing various essential amino acids improve nutritional value, and therefore, such combined proteins are valuable in food production [13,14].
The aim of the study was to evaluate the physicochemical, technological, rheological, and microstructural characteristics of the combined minced meat formulations, optimized in terms of biological value, containing beef trimming, flax-seed flour, and hemp protein, with and without the transglutaminase treatment.

Raw Materials and Ingredients.
Fresh beef trimming (90/10) (66.34% moisture, 17.40% protein, and 9.26% fat) were obtained from rib and round portions 48 h after slaughter. Beef trimmings were packaged in plastic bags and transported in a refrigerator at 4°C to the laboratory of the Food and Biotechnology Department within 2 h for further processing. The pH of the beef trimmings was determined using a portable pH meter (HANNA HI83141). An electrode was inserted to a depth of 5 ± 1 cm, where the values obtained ranged from 5.56 to 5.62.
To obtain the flour, flax seeds were heat-treated at 90°C for 3-5 minutes, defatted by pressing, ground in a colloid mill, and sifted through a sieve with a mesh size of 160 microns.
Hemp protein (9.8% moisture, protein: 56.1% DM, fat: 23.4% DM) was produced from the hemp seeds of the Nadezhda variety at the Medal enterprise (Chelyabinsk, Russia).
To obtain the protein, hemp seeds were heat-treated at 70°C for 10 minutes, defatted by pressing, crushed, and sieved several times. A GMSM-120 industrial mill with sieving equipment was used. The resulting hemp protein and flaxseed flour were stored at a temperature of 15-18°C.

Optimization of the Combined Minced Formulation in
Terms of Biological Value 2.2.1. Optimization of the Minced Formulation. Beef trimming (90/10), hemp protein, and flaxseed flour were chosen as starting components for the combined minced formulation.
The optimization of the combined minced formulation was carried out according to the essential amino acid index (EAAI), which was calculated using the following formula: where a n is the ratio of the amount of each essential amino acid in the investigated protein to its amount in the whole egg protein and n is the amount of EAA (n = 8).
The combined minced formulation was optimized using the SOLVER standard software available in Microsoft Excel (v.2013). The calculation of the formulation consisted of several stages: compiling a data bank and balance equations for the amino acid composition, defining the objective function to optimize formulations, solving the problem using the tool 2 International Journal of Food Science SOLVER, and analysing and selecting a formulation appropriate to the goal. The content of essential amino acids was experimentally detected in the components of the mince via HPLC (high-performance liquid chromatograph) to compile a data bank.

Preparation of Samples for
Hydrolysis. Beef trimmings were chopped in a meat grinder (Fimar 32/RS, Unger, Italy) with a plate having 3 mm diameter holes. The hydrolysis of samples of ground beef trimming, hemp protein, and flaxseed flour was carried out according to the method described by Zinina et al. [15]. The amino acid modification was carried out by phenylisothiocyanate solution in isopropanol to obtain phenylthiohydantoins. The concentration of amino acids in the samples was calculated according to the protein content in grams per 100 g of the product. The protein content of the beef trimming, hemp protein, and flaxseed flour samples was determined using the Kjeldahl method.  The minced meat samples were made in accordance with the formulations obtained by optimization, as presented in Table 1.
Hemp protein and flaxseed flour were hydrated in water at a ratio of 1 : 2, respectively. After uniformly mixing all components with water (20% by weight of raw materials), the combined minces were divided into two parts. Transglutaminase (0.2% by weight of raw materials), previously dissolved in water at a ratio of 1 : 10, was added to one part of each of the minces. A similar amount of water was added to the minces without the addition of transglutaminase. The minced samples from beef trimming were taken as the control sample. Then, all the minced samples were kept at 8°C for 2 h to react with the TG. The following designation for samples was introduced: S-C-minced samples produced from beef trimmings; S-C+TG-the minced samples produced from beef trimmings with added TG; S-1, S-2, S-3, and S-4-the combined minced samples produced according to formulations 1, 2, 3, and 4, respectively; S-1+TG, S-2+TG, S-3+TG, and S-4+TG-the combined minced samples produced according to formulations 1, 2, 3, and 4, respectively, with added TG. Thus, a total of 10 formulations were taken, with five minced samples made for each formulation.

Physicochemical Analyses.
The chemical analyses of the combined minced samples were conducted in triplicate according to the methods described by AOAC [16]: total nitrogen content was assayed by the Kjeldahl method with nitrogen converted into the equivalent protein content using a factor of 6.25 (methods 992.15 and 992.23); moisture was determined according to method 950.46 B; total fat was determined via the Soxhlet method (methods 920.39 C and 960.39). The ash content was determined via the dry ashing method (method 920.153).

Rheological
Measurements. Rheological measurements were conducted using a rotary viscometer (Brookfield R/S SST). The principle of operation is to determine the viscosity and shear stress by measuring the rotation speed of the spindle, which is immersed in the test mince. Viscosity measurement and shear stress range are determined by the spindle speed, the size of the spindle, and the full-scale torque of the calibrated spring. The size of the selected spindle was 30/15, and the full-scale torque was 80%. Viscosity and shear stress values were calculated using a coefficient of 9. Investigations of the rheological properties (ultimate shear stress, effective viscosity) of combined mince were carried out at room temperature (20 ± 1°C).
2.6. Texture Measurements. Deformation indicators were conducted using a texture analyser "Structurometer ST2" (LAB, Quality Laboratory, Russia) by compressing it with an indenter "Cylinder Ø36" (duralumin, mass 42.5 g). The analysis of the mechanical tension induced on a cylindrical indenter was carried out under the following operation mode: contact force (Fc = 7 g), strain rate (Vd = 0:5 mm/s), and continuation of the introduction of the indenter until the effort F max was equal to 500 g. The total plastic and elastic deformations were determined.
All texture measurements were carried out after sample fermentation for 2 h at a temperature of 8 ± 1°C.

Technological Property Measurements
2.7.1. Water Binding Capacity. The water binding capacity (WBC) was determined by the Grau and Hamm method [15]. This method is based on determining the area of a wet spot on filter paper which is formed due to the slight pressure of the minced sample on it. The spot area was determined via a planimeter. The mass fraction of bound moisture was calculated according to the following formula: where X is the mass fraction of bound water in the sample, % total moisture; A is the total mass of moisture in the sample, mg; and B is the area of the wet spot, mm 2 .

Water Holding Capacity.
Water holding capacity (WHC (%)) was determined gravimetrically according to 3 International Journal of Food Science the method described by Zhang et al. [17]. Water holding capacity (%) was calculated using the following formula: where W is the mass of the sample, g; W 1 is the mass of the sample after heating and decanting the supernatant, g; and W 2 is the mass of the sample after centrifuging and removal of the resulting supernatant, g.

Cooking Loss.
Cooking loss (CL (%)) was determined using the gravimetric method, based on measuring the mass of the sample before and after heat treatment at 75°C in the air-o-steam (Rational AQ, Germany) for 10 min. Cooking loss was calculated according to the following formula: where W rs is the weight of raw sample (g) and W cs is the weight of cooked sample (g).

Scanning Electron Microscopy (SEM).
The microstructures of the combined minced samples were examined by scanning in the Scanning Electron Microscope (SEM), JSM 7001F (JEOL, Japan). After freeze-drying the combined minced samples, 10 × 5 × 2 mm plates were cut from the inside of the briquettes and fixed onto the aluminium plate of the sample holder using carbon adhesive tape. The surface of the samples was covered with a layer of electrically conductive material (platinum) with a thickness of about 10 nm. The study was carried out in the registration mode for secondary electrons at a cathode accelerating voltage of 5 kV.
2.9. Statistical Analysis. The analyses were performed in five replicates, each of which was measured three times. The results were expressed as the mean values of the five replicates ± the standard deviation. Probability values of p ≤ 0:05 were taken to indicate statistical significance. The data were analysed via one-way ANOVA and the Tukey test using the free web-based software offered by Assaad et al. [18] 3. Results and Discussion

Optimization of the Combined Minced
Formulation. An important condition in the development of combined products is to achieve the optimal biological value of the protein, as determined by the essential amino acid content.
The formulation of the combined mince was optimized in accordance with essential amino acid content as recommended by the WHO/FAO (World Health Organization/-Food and Agriculture Organization) Expert Consultation [19]. The protein and amino acid content of beef trimming, hemp protein, and flaxseed flour (mg amino acid/100 g product) is presented in Table 2.
The balance equations were based on the experimental data obtained for the content of essential amino acids, as expressed in grams of EAAI per 100 g of protein.
The results of determining the protein content showed significant differences between hemp protein, flaxseed flour, and beef trimming protein. The protein content in the hemp protein was 56.1% (p < 0:05), which is close to the value obtained by Gorissen et al. [20]-51%. The crude protein content in brown and gold flax seeds is 223.25 g/kg dry matter according to Nitrayová et al. [21]. The total protein in flax seed, as found by Sammour [22], was about 20-30% of seed meal, which corresponds to the results we obtained of 39.3% (p < 0:05).
Hemp and flax are protein sources that provide relatively low amounts of lysine (with content of 3.55% and 3.04%, respectively), compared with beef that has a lysine content of more than 8%. This agrees with results obtained by other researchers regarding plant-based and animalbased proteins [20,23].
Methionine and leucine content was low in flaxseed flour (3.23% and 6.06%, respectively) and did not meet the WHO/FAO requirements.
Less pronounced variability was determined between hemp, flax, and beef proteins in valine, isoleucine, and threonine content and met the WHO/FAO requirements.
The optimal ratio of minced components for which EAAI reached 100% can be determined using the SOLVER tool. Indicators of the biological value of the combined minced formulations proposed by the program were automatically calculated ( Table 3).
All selected formulations have high biological value due to the necessary content of all essential amino acids in accordance with WHO/FAO requirements. The EAAI of all formulations was over 100%. Despite the content of limiting amino acids in hemp and flax, it is possible to obtain a balanced minced formulation by combining protein sources of different origin. Gorissen et al. [20] determined that plant-based proteins have relatively low essential amino acid content compared to animal-based proteins. As sources of plant-based protein, the authors studied isolates including oat, lupin, wheat, hemp, microalgae, soy, brown rice, pea, corn, and potato; milk, whey, caseinate, casein, and egg were studied as sources of animal-derived proteins. According to the results of the work, the authors concluded that a balanced combination of different plant-based proteins might provide a highquality protein blend [20]. van Vliet et al. [20,23] noted that the ingestion of several protein sources might provide a more balanced amino acid profile in food.

Physicochemical Analyses.
The results of this physicochemical analysis showed that the protein content of nontreated combined minced groups was found to vary between 13.4% and 14.8%, whereas the protein content of TG-treated samples was significantly increased and determined to be between 14.1 and 15.4% (Table 4). Similar results were obtained by Uran and Yilmaz [24] in the study of burgers with the addition of various concentrations of TG and, indeed, without the addition of TG. The protein content in burgers increased from 13.40 to 14.41% with an increase in the content of TG, whilst in the control sample, the content was 13.19%. Atilgan and Kilic [25] noted that the use of different amounts of binding agents, including TG, generally did not create significant differences in protein levels among different groups of cooked ground meat. Significant differences in the protein content in the formulations with various combinations of plant and animal raw materials were noted. The highest protein values were found for samples S-1, S-2, and S-3 (p < 0:05). A similar dependence was determined by Zając et al. [26], where the addition of hemp seeds to the meat loaves led to a significant increase in protein content.
The moisture content of the combined minced samples produced according to formulations 1, 2, 3, and 4 was 71.6, 72.7, 71.7, and 71.4%, respectively, while the moisture content of samples with TG (S-1+TG, S-2+TG, S-3+TG, and S-4+TG) ranged from 71.3 to 72.5%. It may be noted that the plant components did not significantly affect the moisture content of the minces. According to Atilgan and Kilic [25], the use of microbial TG in ground beef samples did not create significant differences in moisture levels compared to appropriate control groups due to the negative effect of microbial transglutaminase on the moisture level.
The fat content in our study ranged from 7.2 to 9.5% (p < 0:05); statistically significant increases in fat content were found in the nontreated (S-1 and S-3) and TG-treated (S-1+TG and S-3+TG) groups (p < 0:05). Uran and Yilmaz [24] did not notice statistically significant differences in the fat content in the control and other groups containing the enzyme in different concentrations (0.2, 0.6, 0.8, and 1%) in burger production. The fat content in the samples with the high content of plant components (S-1 and S-3) does not differ significantly but was, however, greater than in samples Table 2: Essential amino acids (EAA) and protein content in beef trimming, hemp protein, and flaxseed flour.

Indicators
Beef trimming  5 International Journal of Food Science with a considerable content of beef trimming in formulations (S-2 and S-4). This agrees with the data obtained by Zając et al. [26], who found that the addition of hemp seed slightly increased the fat content in meat loaves.
The ash content of the combined minced samples produced according to formulations 1, 2, 3, and 4 ranged from 3.2 to 3.7%. The ash content in samples S-1 and S-1+TG and S-3 and S-3+TG was significantly increased in comparison with other samples of minced meat (p < 0:05); however, when TG was added to the samples of the combined minces, no significant differences were found. According to Cofrades et al. [27], the quality parameters of chicken steaks supplemented with TG showed no statistical significance compared to control samples in terms of ash content.
The results of proximate analysis obtained by Setiadi and Alisha [28] showed that the addition of transglutaminase enzyme in an animal protein source (duck meat) and vegetable protein (soy powder and texturized soy protein) did not significantly affect its nutrients.
Due to the fact that hemp, protein, and flaxseed flour contain a considerable amount of protein, lipids, and fibre, the addition of these components in an amount more than 18% led to the increase of protein, fat, and ash content in the combined minced samples.

Rheological Measurements.
It was experimentally determined that the rheological properties of minced meat samples without TG treatment changed depending on the proportion and amount of plant raw materials. When the content of the flax flour was increased, the minimum ultimate shear stress and viscosity were observed (S-1 and S-2), while the maximum values for these indicators were achieved in samples containing about 15% hemp protein in the formulation (S-3), as well as in control samples without plant additives (p < 0:05).
The data obtained are related to the fact that flaxseed flour contains a considerable amount of polysaccharides, namely, mucus and pentosans, which are distinguished by a pronounced water-holding and fat-binding ability. The large amount of water and fat in the minced meat matrix leads to a weakening of the bonds between the protein molecules forming the structural network and, as a result, to a decrease in density and viscosity. Hasanvand and Rafe confirmed that flaxseed products contain anionic polysaccharides which have excellent water holding capacity due to their swelling ratio; however, they form a weak gel [29]. The authors investigated the rheological properties of rice bran protein-flaxseed gum complex coacervates and observed that coacervates showed a shear-thinning phenomenon due to the linear reduction of complex viscosity by increasing frequency [30].
The ultimate shear stress and viscosity are related to the strength of intermolecular interactions in the protein-gel matrix, while the change of these indicators is related to the stability of the matrix. In the present study, the S-4 and S-C samples showed stronger intermolecular interactions and a more stable matrix. The addition of hemp might improve the formation of a homogeneous network between the meat protein and hemp protein molecules, hence producing a uniform matrix in the combined food system with improved structural strength and stability (Figures 1(a) and 1(b)).
When adding TG to the formulation, increases in the ultimate shear stress and viscosity were determined for all minced meat samples. Moreover, the TG-assisted crosslinking reaction most affected the samples with the lowest rheological properties. Thus, the ultimate shear stress in samples S-1 and S-4 increased by 44.6 and 26.7%, respectively (p < 0:05), while for the control sample and sample S-3, the changes were 11 and 9.9%, respectively. Therefore, the effect of TG on the formation of additional bonds between plant and animal proteins resulted in a more dense and monolithic structure (Figure 1(a)).

Texture Measurements.
The variation of the deformation characteristics of combined minced samples could be correlated with the results of the viscosity and critical shear stress measurements. Thus, the largest total, plastic, and elastic deformations were found in samples with the lowest shear stress and minimal viscosity (Figure 2). The combined mince, containing more than 8% flax flour, had a more plastic structure and the lowest modulus of elasticity (total and plastic deformation for S-1 and S-2 were 3.9-4.3 and 2.1-1.9 mm, respectively). At the same time, the mince that included about 14% hemp protein (S-3) and the mince containing a more than 87% meat component (S-4) were identical to the control samples in terms of deformation and elasticity. Certainly, in TG-treated mince, the plasticity of the samples reduced while the density and elasticity increased.

Technological Indicator Measurements.
In the combined minces, WBC was characterized by values between 76.7 and 78.8% and WHC by values from 48.5 to 52.5%, without significant variation immediately after their preparation (fermentation period 0 hours). During the first two hours of fermentation, an increase in WBC and WHC was observed for all samples without TG, whilst for samples S-1 and S-2 containing the highest concentration of flaxseed flour, the magnitudes of the indicators increased significantly by 7.3-4.9% (WBC) and 10.4-5.2% (WHC), respectively, relative to the control (p < 0:05) ( Table 5).
For samples S-1+TG, S-2+TG, and S-3+TG, the WBC and WHC values increased significantly by 12.2-18.9% and 19.0-21.4%, respectively, after a 2 h period of TG-assisted cross-linking reaction, relative to the initial values for the same samples. The TG leads to the formation of covalent bonds between the plant and meat protein molecules that allowed water to be retained within the meat matrix under external influence, effectively and positively reducing the cooking loss. The smallest cooking losses were found for the combined minced samples characterized by the highest WBC and WHC values (S-1, S-2, and S-3). A significant decrease in CL was found for all TG-treated samples (p < 0:05). A similar dependence was noted by Duarte et al. [33], where the cross-links formed between the plant proteins by the action of TG greatly influenced the functional characteristics of the products, determining the technological and rheological properties of these systems such as stability, elasticity, and water adsorption. Zając et al. [26] also noted slightly decreased cooking losses in the meat loaves produced with added hemp ingredients.
Thus, TG treatment for 2 hours contributed to the formation of optimal technological properties of combined mince.   The reduction in the amount of plant components in samples S-4 and S-4+TG contributed to the formation of a more compact and dens structure of minced meat, in which individual fragments are difficult to distinguish (Figures 3(d) and 3(d-TG)).
The samples of minced meat obtained without using the TG had a more open structure and greater porosity than the samples of combined mince with TG. Aksoy et al. [35] noted that the porosity and structure of freeze-dried minced meat samples depend on the temperature and drying technique. As seen in the microphotographs, the structure is more compact and denser in the combined minced samples with added TG in agreement with the results of the rheological measurements.
Li et al. [36] also reported about a more compact network of minced mixtures (pork/fish) after addition of 0.4% TG. Mojarrad et al. [37] proved that TG effects on the firmness and provided more cross-linked intermolecular gel structures at high temperatures. Gels containing MTGase supplied a stronger and denser protein network which was formed by cross-linking a network of starch and proteins [37]. The intermolecular bonds between proteins described by Uran and Yilmaz [24] as the G-L (ε (γ-glutamil)-lisil) connections in burger samples are found in combined minced samples with the addition of TG.

Conclusion
The research results have demonstrated the effectiveness of using transglutaminase in the composition of combined minced meat with beef trimming, flaxseed flour, and hemp protein for the formation of a homogeneous and dense system with the necessary technological and rheological properties. The combination of raw materials of both plant and animal origin made it possible to obtain combined minces with optimal amino acid composition of high nutritional value.
The rheological properties of minced meat samples changed depending on the proportion and amount of plant raw materials. The combined mince, containing more than 8% flax flour, had a more plastic structure and the lowest modulus of elasticity. The formulations of mince containing about 14% hemp protein or more than 87% meat component were characterized by optimal values of deformation and elasticity. When adding TG to the formulation, increase in density and elasticity, as well as growth in ultimate shear stress and viscosity, was determined for all combined minced samples.
After two hours of fermentation, an increase in water binding capacity and water holding capacity was observed for all samples. A significant decrease in cooking loss was found for all TG-treated samples. The microstructure of the TG-treated combined minced samples was more compact and dense which corresponded to the results of the rheological measurements.

TG:
Transglutaminase DM: Dry matter EAA: Essential amino acid WBC: Water binding capacity WHC: Water holding capacity CL: Cooking loss.

Data Availability
All the experimental data used to support the findings of this study are available from the corresponding author upon request.


extfieldsofstudy: Medicine Biology

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Arbuscular mycorrhizal fungi composition in soils of switchgrass harvested for bioenergy under 1 alternate nitrogen management

Learning more about the biodiversity and composition of arbuscular mycorrhizal fungi (AMF) 18 under alternative agricultural management scenarios may be important to the sustainable 19 intensification of switchgrass grown as a bioenergy crop. Using PacBio single-molecule 20 sequencing and taxonomic resolution to the level of amplicon sequence variant (ASV), we 21 assessed the effects of nitrogen amendment on AMF associating with switchgrass and explored 22 relationships between AMF and switchgrass yield across three sites of varying productivity in biodiversity our GS24 , studies treatment paired

2 Wisconsin. Nitrogen amendment had little effect on AMF diversity metrics or community 24 composition. While AMF ASV diversity was not correlated with switchgrass yield, AMF family 25 richness and switchgrass yield had a strong, positive relationship at one of our three sites. Each 26 of our sites was dominated by unique ASVs of the species Paraglomus brasilianum, indicating 27 regional segregation of AMF at the intraspecific level. Our molecular biodiversity survey 28 identified putative core members of the switchgrass microbiome as well as novel clades of AMF, 29 especially in the order Paraglomerales and the genus Nanoglomus. Furthermore, our phylogenies 30 unite the unknown, cosmopolitan, soil-inhabiting clade GS24 with Pervetustaceae, an enigmatic 31 family prevalent in stressful environments. Future studies should isolate and characterize the 32 novel genetic diversity found in switchgrass agroecosystems and explore potential yield benefits 33 of AMF richness. 34 to characterize AMF, which fail to detect certain clades while masking the extensive functional 92 diversity present among AMF after over 500 million years of evolution (28). For example, AMF 93 staining procedures to measure root colonization and nutrient-transfer structures do not detect 94 AMF in the order Paraglomerales, which have weakly staining or completely non-staining 95 tissues (29-30). Furthermore, morphological metrics have no inherent meaning for plant 96 performance given that AMF species exist along a symbiotic spectrum ranging from mutualism 97 to parasitism (31-32). In fact, alternative strains within a given AMF species can result in 98 completely different outcomes for plant performance (33-35). In addition, the 16:1ω5cis fatty 99 acid biomarker commonly used to measure AMF abundance in soils (36-37) is only present at 100 miniscule quantities or is completely absent from species in Paraglomeraceae and 101 Gigasporaceae (38). On the other hand, fatty acid indicators for these two families (e.g., 102 16:1ω7cis, 18:1ω9cis, 20:1ω9cis) are typically treated as biomarkers for saprotrophic fungi and 103 gram-negative bacteria, conflating the abundance of multiple guilds (38-40). Finally, AMF 104 metabarcoding of the small subunit ribosomal DNA (rDNA) gene region with short-read 105 sequencing platforms does not discriminate AMF to the species level (27, 41-42). This problem 106 is compounded when operational taxonomic units are clustered at 97% similaritya 107 conservative threshold and arbitrary proxy for species-level delimitation of filamentous fungi 108 (43)(44). 109 In the context of a long-term, replicated bioenergy cropping systems experiment, we 110 conducted a metabarcoding study of AMF using PacBio Sequel Single Molecule, Real-Time 111 6 highly variable regions of rDNA for an unprecedented opportunity to investigate AMF at a fine 115 taxonomic resolution (42). ASVs are exact, error-free sequences and thus serve as biologically 116 meaningful representatives of AMF genotypes, revealing intraspecific variation and strains of 117 interest for bioprospecting (9, 43). To our knowledge, this study is the first to employ PacBio 118 Sequel SMRT Sequencing with ASVs in the analysis of AMF communities and one of only a 119 handful to use PacBio for AMF metabarcoding (18,41,(45)(46)(47). We asked the following 120 questions: Does long-term N amendment affect AMF ASV diversity or community structure in 121 Wisconsin switchgrass agroecosystems? Does AMF diversity explain variation in switchgrass 122 yield? We hypothesized that N amendment would decrease AMF diversity (27,48) and that 123 AMF diversity would be correlated with switchgrass yield because of its positive linkage to 124 ecosystem functioning (3). 125 126 Methods 127

Sites and experimental design 128
The US Department of Energy's Great Lakes Bioenergy Research Center designed a 129 Marginal Lands Experiment to test the efficacy of growing perennial biofuel feedstock crops on 130 abandoned agricultural fields or land deemed unsuitable for high-productivity agriculture. This 131 study focused on three Wisconsin sites: Rhinelander in northern Wisconsin, Hancock in the 132 central part of the state, and Oregon in the south (Fig. S1). The coordinates, soil texture, and 133 mean soil physicochemical properties of the three sites are presented in Table S1; for a 134 comprehensive assessment of the soils at each site, see Kasmerchak and Schaetzl (49)

Taxonomy and phylogeny of ASVs recovered from switchgrass agroecosystems 261
A total of 190,700 high-quality, non-chimeric amplicons clustered into 436 ASVs (for a 262 complete breakdown of the number of sequences remaining after each bioinformatics processing 263 step, see Table S2). Rarefaction curves of ASV accumulation versus sample size reached a clear 264 asymptote for every experimental unit, indicating relatively complete sampling of AMF diversity 265 in taxonomic assignment showcases both the biological (e.g., myriad undescribed taxa in the 285 Nanoglomus plukenetiae sensu lato clade) and artificial (e.g., incomplete data curation of 286 Paraglomerales) issues that hinder accurate identification of AMF environmental sequences 287 using even curated reference databases. 288 A few clades stood out for their high sequence abundance or consistent presence across 289 the experimental units, which suggests a role in the core microbiome of switchgrass ( Fig. 2; 76). 290 The previously mentioned clade encompassing Nanoglomus plukenetiae included ASVs with 291 some of the greatest absolute abundances (ASVs 1, 4, and 9). While no ASV belonging to this 292 clade occurred at more than one site, representatives of the Nanoglomus plukenetiae sensu lato 293 clade were present in both N-amended and control plots at all three sites (Figs. S3-S4). In total, 294 64 of the 436 ASVs (~15%) belonged to this diverse but poorly understood clade, which may 295 represent various undescribed species in the genus Nanoglomus or new genera within 296 on November 4, 2020 by guest http://aem.asm.org/

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Glomeraceae. Within Paraglomerales, 73 ASVs (~17%) had high LSU sequence similarity to 297 Paraglomus brasilianum and 51 more (~12%) to Paraglomus laccatum. Since ASVs belonging 298 to these species also occurred at all three sites (Figs. S3-S4), Paraglomus spp. appear to be 299 important members of the core switchgrass microbiome as well. Of the 436 ASVs recovered in 300 this study, only three ASVs occurred at all three sites, two of which belonged to Paraglomus 301 brasilianum (ASVs 8 and 27); the last -ASV 78belonged to Microdominikia litorea, another 302 potential member of the core switchgrass microbiome (Fig. 2). 303 In addition to the Nanoglomus plukenetiae sensu lato clade, some of the less-abundant 304 clades identified in this study might be undescribed lineages of AMF. Our phylogenetic analysis 305 showed that the unknown, worldwide, soil-inhabiting clade deemed GS24 (77) corresponds to 306 the family Pervetustaceae in Paraglomerales, which contains one species, Pervetustus simplex, 307 described in 2017 from the deserts of Oman (29). We recovered seven ASVs in three clades 308 belonging to this enigmatic family, two of which were well-supported (Fig. 3). These ASVs were 309 most prevalent in the sandy soils of our Hancock site, indicating they may confer some degree of 310 stress tolerance to switchgrass under water-limiting or nutrient-poor conditions (76). Finally, two 311 ASVs -ASVs 187 and 427were phylogenetically resolved as basal to the rest of 312 Paraglomeraceae, suggesting a new genus or a new family in Paraglomerales might be 313 necessary to accommodate this newfound genetic diversity (Fig. 3). 314

Relationship between soil variables and switchgrass yield, and indicator taxa 326
No metric of AMF ASV diversity was a significant predictor of switchgrass yield at any 327 site in linear mixed effects regression models including N treatment. At the family level, AMF 328 richness was a marginally significant predictor of yield at Oregon (F 1,4.92 = 21.3, p = 0.006, R c 2 = 329 0.88); every additional family represented in the AMF community at Oregon was associated with 330 an increase in 0.61 Mg ha -1 of switchgrass yield (Fig. 7). There was also a significant 331 relationship between switchgrass yield and cation exchange capacity at Hancock ( information exists on other taxa prevalent in our study (e.g., Paraglomerales), limiting our ability 391 to determine whether community structure in relation to soil N was a function of morphology 392

Positive correlation trend between yield and AMF family richness at most productive site 393
We found no universal relationships between AMF metrics and switchgrass yield at the 394 ASV level. Indeed, improved taxonomic resolution may not allow one to detect relationships 395 between AMF and plant productivity if fine-scale taxonomic groupings of AMF are not good 396 proxies for functional trait differences. However, at the family level in our most productive site 397 (Oregon), we detected a relationship between switchgrass yield and AMF family richness. Along 398 with N treatment and plot, these variables could account for 88% of the variation in switchgrass 399 yield. In another bioenergy study on productive soils, Emery et al.

Switchgrass soil microbiome dominated by poorly studied taxa 425
Our understanding of AMF is biased towards those that are most prevalent in disturbed 426 habitats (90). Concerted efforts to understand uncultured fungi in less disturbed environments 427 could benefit perennial agriculture as a whole given that these agroecosystems might harbor 428 AMF communities that are more similar to non-agricultural ecosystems. In addition to the 429 numerous undescribed clades identified in this study, little is known about the ecology of the 430 putative core AMF of the switchgrass microbiome.


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Field Validation of on STCR Based Fertilizer Recommendations in Paddy on Alfisols of Nagarkurnool District, Telangana State

On farm trails were conducted at six different locations across three villages in Nagarkurnool district of Telangana during Rabi 2017 and 2018 to evaluate the soil test crop response (STCR) based fertilizer prescription for paddy on Alfisols. The treatments include farmers fertiliser practice, recommended dose of fertilizers (RDF) and STCR based fertilizer recommendations for an yield target of 70 q ha -1 . The N, P and K fertilizers for yield target was calculated based on the initial soil test values of the respective locations. The results showed that, against the paddy yield target of 70 q ha -1 , the grain yield at six locations varied from 69.08 to 72.49 q ha -1 with a mean of 71.15 q ha -1 . With respect to farmers practice of fertiliser recommendations, the yield at ten locations varied from 66.18 to 69.82 q ha -1 with a mean yield of 67.92 q ha -1 . The grain yield recorded with STCR recommendations were higher in producing grain yield as compared to RDF and farmers practice of fertiliser recommendations. Adoption of STCR based fertilizer recommendations for paddy was useful to the farmers for saving of P fertilizers from current recommended dose by 25 kg P2O5 ha -1 (42 % of RDP) and saving of cost of P fertilizers applied per hectare to the extent of Rs.1250/-. The treatment of targeted yield found most economic treatment as compare to farmer practices and general recommendation.

INTRODUCTION
Rice the staple food of more than half of the population of the world, is an important target to provide food security and livelihoods for millions. Fertilizer is one of the costliest inputs in agriculture and the use of the right amount of fertilizer is fundamental for farm profitability and environmental protection (Kimetu et al., 2004). Soil test based fertilizer prescription eliminates over or under usage of fertilizer inputs there by increasing the fertilizer use efficiency and yield of crops.
The fertilizer prescription equations developed by the AICRP centre of the project on STCR for different crops of different agro-ecological situations in different states. Substitution of soil test values and target yield in the prescription equations gives us that particular fertilizer nutrient recommendation which need to be converted in to fertilizer quantity to be applied. These fertilizer target yield equations will not only help in saving of fertilizers and improving the economy but also help in improvement of soil health.
The treatment with N application based STCR realized the target yield (Ramesh & Chandrashekaran, 2007). Srinivasan and Angayarkanni (2008) reported that application of fertilizers based on STCR equation will result in buildup of nutrients in the soil. Soil test based application of plant nutrients helps to realize higher response ratio and benefit: cost ratio as the nutrients are applied in proportion to the magnitude of the deficiency of a particular nutrient and the correction of the nutrients imbalance in soil helps to harness the synergistic effects of balanced fertilization (Rao & Srivastava, 2000). Hence the present investigation was undertaken where STCR approach of fertilizer application was compared with farmers practice and recommended dose of fertilizers application. In this contest, STCR based target yield approach will help to provide the required amount of nutrient to the crop and increases the crop uptake and in turn higher yield can be expected.

MATERIALS AND METHODS
On farm trails were conducted at six different locations across three villages in Nagarkurnool district of Telangana during Rabi 2017 and 2018 to evaluate the soil test crop response (STCR) based fertilizer prescription for paddy on Alfisols. The fertilizer prescription equation developed for desired yield target of paddy (T = 70 q ha -1 ) for Southern Telangana soil series are furnished here.
Where, FN, FP 2 O 5 and FK 2 O are fertilizer N, P 2 O and K 2 O in kg ha -1 respectively. T is the yield targeted in q ha -1 ; SN, SP and SK are soil available N, P and K in kg ha -1 respectively. The treatments include T1: Farmers Fertilizer Practice, T2: Recommended Dose of Fertilizers (RDF) and T3: Soil Test Crop Response (STCR) based fertilizer dose for an yield target of 70 q ha -1 . The test crop paddy variety Telangana Sona (RNR 15048). BCR (B: C ratio) was worked out based on the standard procedure (Gittinger, 1982). The cultivation practices were carried out periodically and the grain and straw yield was recorded at harvest.

Initial available soil nutrient status
Initial soil samples were collected in each location and analysed for alkaline KMnO 4 -N (Subbiah & Asija, 1956), Olsen-P (Olsen et al., 1954) and NH4OAc-K (Hanway & Heidal, 1952). Initial soil samples were collected in each location and analyzed for available status of nutrients. The soils across 6 locations were neutral in reaction to non-saline in nature. Available N, P and K were low, high and medium to high in status ranging from 188 to 232, 31 to 46 and 279 to 348 kg ha -1 , respectively (Table 1). The available status of nutrients was used to compute fertiliser doses for paddy through adjustment equations using basic data that had earlier been generated from fertility gradient field experiments for paddy. The detailed procedure has been described by Valayutham et al (1985 Fig.1). Recommended dose of fertilizer application for paddy in Telangana region is 100-50-40 kg N-P 2 O 5 -K 2 O ha -1 .

RESULTS AND DISCUSSION
The fertilizer application as per the STCR equation with yield target 70 q ha -1 to paddy crop for validation were achieved the desired targeted yield ( Table 2). The yield level that could be achievable with 100 % recommended dose (120-60-40 kg N-P 2 O 5 -K 2 O ha -1 ) to paddy crop can be obtained with a lower dose of 130-35-36 kg N-P 2 O 5 -K 2 O ha -1 to the same crop and thus saving 25 kg cost of P input without sacrificing the yield of rice crop. The grain yield under STCR recommendations at six locations varied from 69.08 to 72.49 kg ha -1 with a mean of 71.15 q ha -1 . The variation in yield obtained from the targeted ones ranged from -1.31 to +1.64. The fertiliser application as per the STCR equation with yield target 70 q ha -1 to paddy crop for validation were achieved the targeted yield. With respect to farmers practice of fertilizer recommendations, the yield at ten locations varied from 66.18 to 69.82 q ha -1 with a mean yield of 67.92 q ha -1 . The grain yield recorded with STCR recommendations were higher in producing grain yield as compared farmers practice of fertilizer recommendations (Table 3). Singh et al. (2014) reported that targeted yield of rice was achieved by using the plant nutrients on the basis of targeted yield concept (soil test crop response technology). The present results were in accordance with the study conducted by Singh et al. (2017) and revealed that among the treatments, STCR recorded relatively higher yield, beneit: cost ratio and per cent achievement. Suresh and Santhi (2018) validated STCR equation for hybrid maize and reported that, STCR based fertilizer recommendations with the targeted yield has been achieved within +/-10 per cent variation proving the validity of the equations. The results of the validation experiment on paddy clearly indicated that the per cent achievement was below 10 per cent variation at all the locations for validation was achieved the targeted yield of paddy crop.
The benefit cost ratio of the treatments was estimated using the cost of input and value of output. Economics of fertilizer application based on targeted concept gave benefit cost ratio varying from 2.27 to 2.36 with a mean value of 2.31. The corresponding value for farmers fertilizer practice ranged from 1.96 to 2.02 with a mean value of 2.00 (Table 3). The results are confirming that, the treatment of targeted yield found most economic treatment as compare to farmer practices and general recommendation. Similar results were reported by Singh et al. (2015). The gross and net returns in T1 were Rs. 1,07,988/-and Rs.53,952/-, respectively (Table 4) where as in T3 the higher gross and net returns were obtained (Rs.1,13,123/-and Rs.64,160, respectively). Chaubey et al. (2015) reported that the net returns from the improved practice (STCR technology) were substantially higher than the farmers' practice (GRD).


extfieldsofstudy: Biology Medicine

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Genetic diversity and population genetic structure of six dromedary camel (camelus dromedarius) populations in Saudi Arabia.

Camels are an integral and essential component of the Saudi Arabian heritage. The genetic diversity and population genetic structure of dromedary camels are poorly documented in Saudi Arabia so this study was carried out to investigate the genetic diversity of both local and exotic camel breeds. The genetic diversity was evaluated within and among camel populations using 21 microsatellite loci. Hair and blood samples were collected from 296 unrelated animals representing 4 different local breeds, namely Majaheem (MG), Maghateer (MJ), Sofr (SO), and Shaul (SH), and two exotic breeds namely Sawahli (SL) and Somali (SU). Nineteen out of 21 microsatellite loci generated multi-locus fingerprints for the studied camel individuals, with an average of 13.3 alleles per locus. Based on the genetic analyses, the camels were divided into two groups: one contained the Saudi indigenous populations (MG, MJ, SH and SO) and the other contained the non-Saudi ones (SU and SL). There was very little gene flow occurring between the two groups. The African origin of SU and SL breeds may explain their close genetic relationship. It is anticipated that the genetic diversity assessment is important to preserve local camel genetic resources and develop future breeding programs to improve camel productivity.

Introduction
Arabian camels belong to the genus Camelus that contains only two species, one-humped (C. dromdarius) and two-humped (C. bacterianus) camels. Arabian camels were domesticated in southern Arabia thousand years before they were known in the north part of Arabia (Wardeh, 1989). Camels are unique animals in many aspects and cannot be compared with other farm animals in their physiological responses or adaptation to arid environment (Schwartz and Dioli, 1992;Sweet, 1965). Due to the unique characteristics of camel, the work on evaluation of breeds/strains is an advantage. The camels of Saudi Arabia are mainly dromedaries, the one-humped camels. They are an integral and notable compo-nent of the Kingdom heritage, a symbol of its nomadic traditions. Saudi Arabian rapid modernization has moved camel caravans to be a part of the past, although the animal still provides transportation for some bedouin in remote areas. The total population of dromedary is estimated to be around 1.6 million camels within the Arabian Peninsula, about 53% found in Saudi Arabia (FAO, 2011). Genetic studies on camels are scarce; therefore, the genetic diversity assessment of Saudi Arabian camels is important to preserve their genetic resources and to improve their breeding programs. The development of molecular biology during the past decades has offered new tools in many biotechnological disciplines including livestock genetics and animal breeding (Hines, 1999;Walsh, 2001). These biotechnological tools can be applied specifically to improve camels' productivity, preserve their genetic resources and develop successful breeding programs. Numerous studies on the genetic relationships among and within farm animals breeds have been reported using different genetic markers (Mannen et al., 1998;Ming et al., 2017;Hedayat-Evrigh et al., 2018). Among these genetic markers, microsatellites have been extensively used for assessing the genetic diversity and relationships within and among closely related farm animal populations (Geng et al., 2003;Li et al., 2004;Yang et al., 2004). Microsatellites are valuable genetic markers due to their dense distribution in genomes, easy to develop and inexpensive. Microsatellite analysis simply measures the nucleotide length of different alleles at a particular locus across different individuals. Allelic diversity represents how many different alleles are present throughout a population for each locus. Both Allelic diversity and heterozygosity can provide a direct measurement of genetic diversity in animal populations. As in other domestic animal species, microsatellites in camels are highly informative polymorphic markers and becoming the technique of choice forinvestigating genetic diversity and population studies (Al-Swailem et al., 2009;Mburu et al., 2003;Schulz et al., 2005). In Saudi Arabia, genetic studies on camels are scarce and the extent to which dromedary populations are genetically differentiated is poorly documented. The present study was, therefore, carried out to (1) determine the genetic diversity of four local populations and two exotic ones, existing in Saudi Arabia and to (2) infer the genetic relationship within and among them. It is anticipated that the assessment of genetic diversity of Saudi camels is important to preserve their genetic resources and to develop future breeding programs to improve camel productivity.

Animal resources and DNA isolation
Five raising-camels regions, covering East, West, North, South and middle localities in Saudi Arabia, were visited for sampling camels. Hair and blood samples were collected from 296 camels representing 4 indigenous breeds, 50 Magaheem (MG), 50 Maghateer (MJ), 50 Sofr (SO) and 50 Shaul (SH), in addition to two exotic ones, 48 Sawahli (SL) and 48 Somali (SU). The collected samples were kept in À20°C until use. Information about camel breeds, e.g. history of sampled populations, were also recorded. DNA was extracted using the QIAgen DNeasy blood and tissue kit (Hildane, Germany) following the manufacturer's instructions.

Polymerase chain reaction (PCR) and fragment analysis
Twenty one microsatellite primer-pairs (FAO, 2000) were used to genotype the 296 camels (Table 1). To test the usability of the 21 microsatellites in genotyping sampled camels, pooled DNA preparations from apparently distant populations were firstly tested. PCR gradients were also used to obtain the optimum annealing temperatures for every primer-pair of each microsatellite marker. The amplification was performed using the Gene Amp PCR System 9700 thermocycler (Applied Biosystems, Warrington, UK) according to Mahmoud et al. (2012). The amplification protocol included an initial denaturation step for 2 min at 94°C, followed by 35 cycles at 94°C for 30 s, 50-60°C annealing temperature (depending on the primer-pair used) for 30 s and 72°C for 30 s. The final step of the amplification protocol was an extension step at 72°C for 5 min. Agarose gels were used to check the amplification reproducibility from DNA preparations. The amplified PCR products were multiplexed based on their fluorescent dye and sizes. Each 0.5 ml of multiplexed PCR products were mixed with 9.25 ml of HiDi formamide and 0.25 ml of GeneScan Ò LIZ standard Applied Biosystems. This mixture was immediately denatured at 95°C for five minutes and chilled on ice for five minutes. Two microliters of the previous mixture were loaded into DNA capillary ABI Prism Ò 3500 Genetic Analyzer. The used fluorescent dyes were FAM, NED, VIC and PET. The raw data were collected using genetic analyzer data collection software version 3.0.

Genetic analyses
The basic parameters for each locus and populations, including allele frequencies, observed number of alleles (Na), effective number of alleles (Ne), observed (Ho) and expected (He) heterozygosities values were calculated using Cervus version 3.0.3 (Kalinowski et al., 2007). Wright's F-statistics (F IS , F ST , and F IT ) within and among the camel populations were calculated by using GenePop version 4.0.10 (Raymond, 1995). Deviations from Hardy-Weinburg equilibrium were also calculated by using the GenePop. Factorial correspondence analysis (AFC) 2D plots were performed on a table of allele frequencies using GENETIX 4.05.2 (Belkhir et al., 2004) to infer population differentiation (Jombart et al., 2009). Population structure was estimated using Structure v.2.3 (Pritchard et al., 2000) through the population admixture model. Simulations were run with Markov Chain Monte Carlo (MCMC) of 2.2 Â 10 6 iterations after a burn-in of 2 Â 10 5 . Six independent simulations for different K values (1-8) were performed to identify the most probable clustering solution through examining the modal distribution of DeltaK (DK) (Evanno et al., 2005). The results from multiple runs for each K were concatenated by clump (Jakobsson and Rosenberg, 2007).

Results and discussion
The present study included 296 individuals of dromedary camels belonging to six populations: MG, MJ, SO, SH, SL and SU. The camel individuals were genotyped using 21 microsatellite loci ( Table 1). Out of the 21 microsatellite primer-pairs, 19 successfully generated scorable polymorphic fingerprints from the sampled animals. The VOLP67 and CMS25 primer-pairs did not produce any PCR products. The total number of alleles (Na), mean effective number of alleles (Ne), observed (Ho) and expected (He) heterozygosities were estimated for the six populations. The number of alleles per locus ranged from 7 to 23, with an average of 13.3 alleles per locus (Table 2). This range was comparable with that observed by  in Australian dromedary camels with an average of 13.18. However, Mehta et al. (2007), Al-Swailem et al. (2009), Nolte (2003 and Schulz et al. (2010) reported an average of 10.7, 3.81, 10.3 and 10.3 alleles per locus in Indian, Saudi, South African, Canarian camel populations, respectively.
The total number of alleles generated from the sampled camels using the 19 microsatellite primer-pairs was 253 alleles. The observed alleles varied within SL, SU, MG, MJ, SH and SO populations, with a total numbers of 158, 139, 161, 155, 168 and 162, respectively (Table 2). It is proposed that the number of alleles in a population is highly dependent on the sample size and the presence of unique alleles with low frequencies (Kalinowski, 2004). Generally, as the sample size increases, the number of alleles increases. Al-Swailem et al. (2009) reported a total number of 61 alleles generated from 99 animals using 20 microsatellite primerpairs. The other previous camel studies varied in the total number of alleles where different numbers of individuals and microsatellites were used Nolte, 2003;Schulz et al., 2010;Spencer and Woolnough, 2010).
In this study, CVRL06 was the lowest polymorphic microsatellite locus with seven alleles detected in the studied camel populations (Table 2). Vijh et al. (2007) reported that CVRL06, amongst 23 loci, was also the lowest polymorphic locus with 3 alleles in Indian Bikaneri breed. Although Mariasegaram et al. (2002) found that CVRL08 had the lowest number of alleles; they recorded 3 alleles of CVRL06 in dromedary camels. Moreover, 3 and 4 alleles of CVRL06 were detected in Tunisian Kebili and Medenine camel populations, respectively (Ahmed et al., 2010). On the other hand, YWLL08 was the most polymorphic locus with 23 alleles (Table 2). Many studies reported different numbers of YWLL08 alleles.    found 24 alleles of YWLL08 in dromedary racing camels. Other studies reported variable number of YWLL08 alleles ranging from 9 to 20 in different camel populations, e.g. Indian Bikaneri and Australian camels (Mahmoud et al., 2012;Vijh et al., 2007;Spencer and Woolnough, 2010;Hashim et al., 2014;Eltanany et al., 2015;Nolte, 2003;Schulz et al., 2010). According to Bishop et al. (1994), there is a positive relationship between the number of dinucleotide repeats and the number of alleles at a given locus, explaining why YWLL08 (size range 133-180 bp) has more detected alleles than CVRL06 (size range 185-205 bp). The heterozygosity level of a microsatellite marker varies from species to species and sometimes among different breeds of the same species (Guichoux et al., 2011). In the present study, the average heterozygosity observed in SL, SU, MG, MJ, SH and SO populations were 0.914, 0.929, 0.765, 0.717, 0.743 and 0.713, respectively (Table 3), whereas their expected values were 0.707, 0.702, 0.700, 0.667, 0.695 and 0.691, respectively (Table 3). Generally, the observed heterozygosity values were higher than expected, with the lowest expected heterozygosity value of 0.667 detected in MJ population. However, the highest value was 0.707 in SL population. The highest observed heterozygosity value was in SU camel population, whereas the lowest value was in SO population. The average observed heterozygosity in the present study (0.713-0.929) was higher than reported by previous studies in Tunisian (0.460), Arabian (0.552) and Australian camel populations (0.455) Nolte, 2003;Kalinowski, 2004). Vijh et al. (2007) observed heterozygosity values of 0.580, 0.570, 0.560 and 0.600 for Bikaneri, Jaisalmeri, Kutchi and Mewari populations, respectively. The study of Al-Swailem et al. (2009) on Saudi camels have dealt with three breeds of camels including; Magaheem, Sofr and Shogeh. They found very little variation between the three breeds and low heterozygosity concluding that the three types are closely related. The preliminary study carried out by Mahmoud et al. (2012) on MG, MJ, SO and SH populations from Riyadh area, Saudi Arabia, showed that the observed heterozygosities ranged from 0.605 to 0.665.
The maximum Ne values of YWLL08 locus were 7. 200, 9.198, 7.764 and 6.803 for SL, SU, MJ and SO populations, respectively. However, Ne in MG Population was 7.123 for CVRL07 locus and 6.649 in SH population for locus CMS50 locus. The means of effective number of alleles were 3.938, 2.944, 3.770, 3.599, 3.774 and 3.590 for SL, SU, MG, MJ, SH and SO populations, respectively. Australian camels displayed an average effective number of alleles of 3.44 whereas Bikaneri camels displayed the highest average effective number of alleles of 4.40 (Mehta et al., 2007;Vijh et al., 2007).
The average F IS , F IT and F ST values were À0.165, À0.090 and 0.063, respectively ( Table 5). The low F IS and F IT values may indicate low level of inbreeding within and among the studied populations, with low genetic differentiation among them. The F ST values indicate that less than 1% of the total genetic variation was explained by a population difference, whereas the remaining 99% corresponding to differences among individuals.
To avoid the standard error of distance estimates, Barker (1994) suggested microsatellite loci with fewer number of alleles (>4) should be removed from the genetic distance studies. In our study, none of the alleles was removed from the genetic distance analysis as the lowest number of alleles per locus was 7. The pairwise genetic distance showed that the Saudi indigenous camel breeds (MG, ML, SO, and SH) are genetically close to each other. The pairwise genetic distances among these four breeds ranged between 0.023 and 0.041 (Table 6). The SL breed showed larger genetic distances with the previous four breeds with genetic distances ranged between 0.296 and 0.337 (Table 6). Interestingly, the exotic Somali SU breed was genetically distant from the Saudi breeds with pairwise genetic distance ranged from 0.373 to 0.411 (Table 6). However, the pairwise genetic distance between SU and SL was 0.082 indicating close genetic relationship and most probably both are recently introduced to the Kingdom. The 2D AFC based on allele frequencies grouped the accessions into two well differentiated clusters. The first principal component (PC) explained 6.31% of the total variance and distinguished between these clusters. The first cluster included all SL and SU individuals and the second one included the remaining individuals from populations MG, MJ, SH and SO. Another way to visualize the eventual groups was to analyze the Arabian C. dromdarius microsatellite dataset using Structure with no prior distribution specified. Structure revealed that K = 3 had the highest DK, and has a strong peak at the ''true" number of clusters, suggesting that the optimal number of K was 3 (Fig. 1A). In this analysis, the colors represent the three different clusters (Fig. 1B). Each bar in the graph represents an animal with its inferred proportion of genome admixture. The proportion of membership of each pre-defined population in each of the 3 clusters was 99.8% to the first cluster (red) for SL and SU breeds. This proportion reached 50% for MG, MJ, SH and SO to the second (green) and the third (blue) clusters, respectively. The bar plot graph shows a unique ancestral genome for both of SL and SU. However, camels from the MG, MJ, SH and SO appear sharing allele frequencies of the green and blue groups. The lowest genetic distance was observed between the second and third populations (d 2-3 = 1%). However, it reached 11% between the first and third populations.

Conclusion
Microsatellite markers were very useful to elucidate the genetic diversity within and among camel populations in Saudi Arabia. Based on the genetic analyses, the camels were divided into two groups: one contained the Saudi indigenous populations (Majaheem, Maghateer, Shaul and Sofr) and the other contained the non-Saudi ones (Somali and Sawahli). There was very little gene flow occurring between the two groups.

Declaration of Competing Interest
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. Fig. 1. Genetic structure of 296 unrelated camels representing six breeds from Saudi Arabia using Structure. (A) A plot of two graphical methods allowing detection of the true value for K. The blue (square) represents the mean of LnP(k) over 8 runs for each K value analyzed the increase of the variance associated to LnP (D) across different K values tested. The red (circle) represents the values of DK calculated, based on the methodology proposed by Evanno et al (2005). (B) A bar plot of individual ancestry proportions for the genetic clusters inferred using STRUCTURE for the optimal value of K estimated (K = 3).


extfieldsofstudy: Chemistry Medicine

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Investigating the Effects of Chinese Cabbage Powder as an Alternative Nitrate Source on Cured Color Development of Ground Pork Sausages

Abstract This study investigated the effects of Chinese cabbage powder as a natural replacement for sodium nitrite on the qualities of alternatively cured pork products. Chinese cabbages grown in Korea were collected and used for preparing hot air dried powder. Different levels of Chinese cabbage powder were added to pork products and evaluated by comparing these products to those with sodium nitrite or a commercially available celery juice powder. The experimental groups included control (100 ppm sodium nitrite added), treatment 1 (0.15% Chinese cabbage powder added), treatment 2 (0.25% Chinese cabbage powder added), treatment 3 (0.35% Chinese cabbage powder added), and treatment 4 (0.4% celery juice powder added). The cooking yields and pH values of treatments 1 to 3 were significantly lower (p<0.05) than the control. However, all of the alternatively cured products were redder (higher CIE a* values; p<0.05) than the control and this result was supported from higher nitrosyl hemochrome, total pigment, and curing efficiency. Furthermore, the inclusion of vegetable powders to these products resulted in considerably less residual nitrite content. However, Chinese cabbage powder (0.25% and 0.35%) was effective in producing alternatively cured meat products with a higher curing efficiency comparable to those of the traditionally cured control or the products with celery juice powder. Therefore, Chinese cabbage powder exhibited the efficacy for use as a natural replacer for alternatively cured meat products.

Introduction
Nitrite, which is essentially used for curing meat, is a typical synthetic additive to replace in processed meat products termed naturally cured meat products. Nitrite is a multifunctional ingredient in cured meat and contributes to the cured meat color and unique flavor, while effectively controlling rancidity by retarding lipid oxidation, and inhibiting the growth of microorganisms, particularly against Clostridium botulinum (Alahakoon et al., 2015;Sebranek, 2009;Sindelar and Houser, 2009; ARTICLE that can serve similarly as a multifunctional ingredient (Alahakoon et al., 2015;Sindelar and Houser, 2009). In order to address this, the meat processing industry seeks to replace synthetic additives with natural sources (Alahakoon et al., 2015).
Novel food technology ideas designed to replace synthetic nitrite have focused on a method for producing naturally cured meat products by adding a vegetable powder with high nitrate content and a starter culture, such as Staphylococcus carnosus, Staphylococcus vitulinus, and Staphylococcus xylosus, possessing nitrate-reducing activity (Sebranek and Bacus, 2007;Terns et al., 2011). Celery juice powder is the most widely used as a natural nitrate source for alternatively cured meat products. However, there is a limit to the amount of celery powder that can be used in product formulation as it may adversely affect the sensory attributes (Sindelar et al., 2007b). Therefore, in order to overcome this problem, some researchers have trialed other types of vegetables with high nitrate content such as white kimchi, red beet, parsley, spinach, and Swiss chard as potential natural sources for nitrite to produce naturally cured meat sausages (Choi et al., 2020;Riel et al., 2017;Riyad et al., 2018;Sucu and Turp, 2018). Riyad et al. (2018) reported that the addition of red beet powder increased the redness of cooked sausages and demonstrated similar lipid oxidation to the nitrite-added control after 4 weeks of storage. However, Riel et al. (2017) found that compared with the traditionally nitrite-cured control, alternatively cured sausages with 0.49% parsley extract powder showed a reduced residual nitrite content (40%) and similar CIE a* values until 21 d of storage. Most of these studies appear to have achieved a successful outcome to replace nitrite because this indirect curing method is used mainly in comminuted meat products rather than the whole muscle products, including hams and jerkies. Since the naturally occurring nitrate is soluble and the starter culture is water insoluble, the results were ineffective in the whole muscle products (Sebranek and Bacus, 2007;Sindelar et al., 2010).
Vegetable concentrates, or powders are advantageous as they can provide high concentrations of nitrate to produce naturally cured products. However, high levels of vegetable products may affect the sensory attributes, and therefore the specific vegetable used should not negatively influence the characteristic flavors and pigments of the cured meats or act as an allergen (Sebranek and Bacus, 2007;Sebranek et al., 2012). Chinese cabbage is a green leafy vegetable and considered a good source for biologically active metabolites such as carotenoids, phenolic acids, flavonoids, and vitamins, which can act as antioxidants (Lee et al., 2015;Seong et al., 2016). Moreover, Chinese cabbage contains high concentrations of naturally occurring nitrate, which makes it a potential candidate for use as a natural alternative for meat products. Reportedly, the average nitrate content in fresh Chinese cabbage was 1,438.5 mg/kg (Kang et al., 2016) or 1,740 mg/kg (Chung et al., 2003) depending on the study. Thus, the nitrate concentration can increase following the drying process and a sufficient amount of nitrate will be supplied to the cured products in powder form (Choi et al., 2020;Sindelar, 2006). Therefore, Chinese cabbage powder was expected to show potential as a new natural alternative for synthetic nitrite as it is not associated with allergens.
However, studies focusing on the compatibility of this ingredient in naturally cured meat sausages have not yet been elucidated. Therefore, this study investigated the effects of the addition levels of Chinese cabbage powder on the quality attributes of alternatively cured pork sausages and evaluated their effects by comparing them with products containing synthetic sodium nitrite or those cured with commercially available celery powder.

The preparation of raw materials
Chinese cabbages grown in five provinces of South Korea (Gyeonggi-do, Gyeongsang-do, Jeolla-do, Chungcheong-do, and Gangwon-do) were purchased, randomly selected (approximately 10 kg/region), and used for manufacturing Chinese cabbage powder in this study. Following removal of the non-edible portion of the cabbages such as the outside leaves and roots, the cabbages were cut (3×5 cm), rinsed in distilled, deionized water, and then drained for 1 h in a strainer. The excess water was further removed by (pressing 10 pumps) using a spinner (OXO Good Grips Salad Spinner, OXO International, Chambersburg, PA, USA). The rinsed Chinese cabbages were homogenized for 4 min using a food cutter (C6 VV, Sirman, Marsango, Italy). The sides of the food cutter were scraped periodically and the sample homogenized for an additional 2 min.
This process was repeated until the sample was homogenized for a total of 10 min (Nuñez de González et al., 2015). The homogenized samples were vacuum-packed in nylon/polyethylene bags (~500 g each) and then stored in a freezer (C110AHB, LG Electronics, Changwon, Korea) at −18℃ prior to hot air drying. Then the frozen samples were removed from the bags and dried at 60℃ in a dryer (EN-FO-3925, Enex Science, Goyang, Korea) for 12 h. The dried samples were pulverized with a blender (51BL30, Waring Commercial, Torrington, CT, USA) for 3 min and screened using a 30 mesh sieve (Test sieve BS0600, Chunggye Sieve, Gunpo, Korea). The Chinese cabbage powder was vacuum-packed in nylon/polyethylene bags and stored in a freezer at −18℃ until further use. The prepared Chinese cabbage powder had a nitrate ion content of 36,863 ppm (equivalent to 50,479 ppm sodium nitrate), a nitrite content of 0.2 ppm, a pH of 5.44, and a moisture content of 6.27%. Celery powder (VegStable 502, Florida Food Products, Eustis, FL, USA) with a nitrate ion content of 23,375 ppm (equivalent to 32,020 ppm sodium nitrate) and a starter culture (CS 299, CHR Hansen, Milwaukee, WI, USA) consisting of S. carnosus, sodium nitrite (S2252, Sigma-Aldrich, St. Louis, MO, USA), and sodium ascorbate (#35268, Acros Organics, Geel, Belgium) were obtained from commercial suppliers.

The manufacturing process of ground pork sausages
The pork ham (M. biceps femoris, M. semitendinosus, and M. semimembranosus) muscles and back fat used in this study were purchased from a local meat processor (Pukyung Pig Farmers Livestock, Kimhae, Korea) at 24-48 h postmortem. The intermuscular fat and visible connective tissues were trimmed from the fresh ham. The lean pork ham was cut into squares of approximately 4-5 cm, vacuum-packaged in nylon/polyethylene film bags, and then stored in a -18℃ freezer until processing within 1 mon. The pork back fat was also prepared similarly. In order to manufacture the ground pork products (total batches of 30 kg per trial), frozen pork meat and back fat were thawed at 2℃-3℃ for 24-36 h prior to processing and then sequentially ground with a chopper (TC-22 Elegant plus, Tre Spade, Torino, Italy) equipped with an 8-mm plate and 3mm plate. The ground pork meat and back fat were randomly separated into five batches (Table 1) as follows: control, 0.01% sodium nitrite; treatment 1, 0.15% Chinese cabbage powder and 0.015% starter culture; treatment 2, 0.25% Chinese cabbage powder and 0.025% starter culture; treatment 3, 0.35% Chinese cabbage powder and 0.035% starter culture; and treatment 4, 0.4% celery juice powder and 0.04% starter culture. In the control samples, the addition of 100 ppm sodium nitrite was considered according to the regulations of The Korea Food Code (Ministry of Food and Drug Safety, 2020) in processed meat (<70 ppm residual nitrite ion). The utilization level of the celery juice powder and starter culture was based on the suppliers' recommendations (Choi et al., 2020;Sindelar et al., 2007b). From the results of the nitrate analysis for Chinese cabbage powder (50,497 ppm nitrate) and celery juice powder (32,020 ppm nitrate), the various addition levels of Chinese cabbage power in treatments 1-3 were selected based on the target nitrate concentration in 0.4% celery juice powder. The ingoing nitrate concentrations from the vegetable powders were calculated as 75.75 ppm, 126.24 ppm, 176.74 ppm, and 128.08 ppm for treatments 1, 2, 3, and 4, respectively. For processing of the control samples, ground pork meat, pork fat, NaCl, dextrose, sodium tripolyphosphate, sodium nitrite, and sodium ascorbate were added to a mixer (5K5SS, Whirlpool, St. Joseph, MI, USA) with the ice/water and then mixed for 10 min (Table 1). For alternatively cured products, Chinese cabbage powder or celery juice powder and starter culture were incorporated in the same ingredients and procedure of the control, but sodium nitrite was not included. Each batch was stuffed into conical tubes (approximately 50 g) using a stuffer (MOD.5/W Deluxe, Tre Spade, Torino, Italy). The stuffed tubes were then centrifuged at 2,000×g for 10 min (Combi R515, Hanil Science Industrial, Incheon, Korea) to remove any air pockets. All tubes were closed with caps and placed on racks in a refrigerator at 10℃ for the control or incubated for 2 h at 40℃ in an incubator (C-IB4, Changshin Science, Pocheon, Korea) for treatments 1 to 4. After incubation, the tubes from each batch were sequentially cooked to an internal temperature of 75℃ in a 90℃ water bath (MaXturdy 45, Daihan Scientific, Wonju, Korea) for approximately 9-12 min depending on the samples. The temperature was monitored using a 4-channel digital thermometer (Tes-1384, Ketech Scientific Instrument, Kaohsiung, Taiwan). Once cooked, the samples were immediately cooled for 20 min on slurry ice and stored at 2℃-3℃ in the dark prior to analysis. Experiments were performed in triplicate.

Moisture content determination
The moisture content of the Chinese cabbage powder was determined using the drying method (AOAC, 2016).

The pH values determination
Five grams of Chinese cabbage powder or cooked meat products were homogenized with 45 mL of distilled water for 1 min in a homogenizer (DI 25 basic, IKA ® -Werke GmbH & Co. KG, Staufen, Germany). The pH values of each sample were measured using a pH meter (Accumet AB150, Thermo Fisher Scientific, Singapore).

Cooking yield determination
The weight of each meat product sample in the conical tube was measured prior to cooking and then again after cooking and cooling to determine the cooking yield using the following equation: Cooking yield (%) = Sample weight after cooking Sample weight before cooking × 100

CIE color measurements
Color measurements were taken using a colorimeter (Chroma Meter CR-400, illuminant C, 2° standard observer; Konica Minolta Sensing, Osaka, Japan) calibrated with a white plate (L* 94.90, a* -0.39, and b* 3.88). The Commission Internationale de l'Eclairage (CIE) L* (lightness), a* (redness), and b* values (yellowness) system was used to determine the color of freshly cut surfaces of each cooked sample immediately after cutting.

Residual nitrite analysis
The nitrate ion (NO 3 − ) and nitrite ion (NO 2 − ) of the homogenized Chinese cabbage prior to drying and the powder following the drying process were determined by the zinc reduction method described by Merino (2009). Results were reported as ppm. The residual nitrite content in the cooked pork sausages was analyzed according to the procedure of the AOAC (2016). A calibration curve was prepared using sodium nitrite (S2252, Sigma-Aldrich, St. Louis, MO, USA), and the residual nitrite content was reported as ppm.

Nitrosyl hemochrome, total pigment, and curing efficiency determination
Nitrosyl hemochrome and total pigment were determined using a method by Hornsey (1956). For nitrosyl hemochrome determination, 10 g of each cooked sample was blended with 40 mL acetone and 3 mL distilled, deionized water using a homogenizer (DI 25 basic, IKA ® -Werke GmbH & Co. KG). The samples were kept in the dark for 15 min and filtered through a Whatman No. 1 filter paper, and then, the absorbance of the filtrate at 540 nm (A 540 ) was determined using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The nitrosyl hemochrome concentration (ppm) was calculated as A 540 ×290. For the total pigment measurement, 10 g of each cooked sample was blended with 40 mL acetone, 1 mL HCl, and 2 mL distilled, deionized water, which was allowed to stand in the dark at 2℃-3℃ for 1 h and then filtered through a Whatman No. 1 filter paper. Absorbance was measured at 640 nm (A 640 ). The total pigment concentration (ppm) was calculated as A 640 ×680. Curing efficiency (%) was calculated using the following equation: Curing efficiency (%) = Nitrosyl hemochrome Total pigment × 100

Statistical analysis
The experiment was replicated three times. Data were statistically analyzed as a randomized block design with five treatments (control and four nitrite-free treatments) using the Proc GLM (general linear model) procedure of the SAS program (SAS, 2012). If significance was determined (p<0.05) in the model, the significance of the means was further separated by Duncan's multiple range test.

Cooking yield, pH values, and CIE color values
The alternatively cured sausages (treatments 1 to 4) produced lower cooking yields (p<0.05) compared to the nitrite-added control (Table 2). However, there were no differences (p>0.05) in cooking yields by Chinese cabbage powder level or the addition of celery juice powder. Kim et al. (2019a) and Krause et al. (2011), who reported that the meat products cured with a natural nitrite source from vegetable juice showed no significant differences in cooking loss compared to the conventionally cured control. However, the effects of vegetable powder on cooking yields in this study are in agreement with Choi et al.
(2020), who found that the naturally cured products with white kimchi powder or celery powder exhibited significantly lower cooking yields than the nitrite-added control.
Naturally cured sausages (treatments 1 to 3) with the Chinese cabbage powder had lower pH values (p<0.05) than the control (Table 2). Moreover, the pH values decreased (p<0.05) further as level of Chinese cabbage powder increased. These decreases in pH might be related directly to the vegetable powder or caused by the lactic acid produced by the starter culture during incubation (Gøtterup et al., 2008;Sebranek and Bacus, 2007;Terns et al., 2011). Since the Chinese cabbage powder used in this study had a pH of 5.44, the powder could lower the pH of treatments 1 to 3. However, the pH values of treatment 4 were similar (p>0.05) to that of the control. This result was consistent with the findings of Sindelar et al. (2007b), who found that the addition of celery juice powder at various levels to hams did not change the pH of the products compared to the nitrite-added control.
No differences (p>0.05) in CIE L* values were observed between the control and the other treatments (Table 2) also found no differences in CIE L* values between the sodium nitrite-added control and beef sausages cured with celery, parsley, or spinach powder. Furthermore, the alternatively cured sausages (treatments 1 to 4) exhibited higher CIE a* values than the control; however, these differences were not for the significant (p>0.05) for the CIE a* values by any Chinese cabbage powder level used in treatments 1 to 3 (Table 2). Similar results were reported by Kim et al. (2019a), who found that when fermented spinach juice was added into pork loin along with organic acids, CIE a* values were higher than in the control marinated with nitrite and ascorbic acid. Similarly, Sucu and Turp (2018) reported that the inclusion of beetroot powder as a nitrite alternative resulted in an increase in redness (higher CIE a* values) in fermented beef sausages. In this study, treatment 2 and treatment 4 provided equivalent nitrates from vegetable powder, while treatment 1 had 40% lower nitrate than the two treatments. Interestingly, the CIE a* values for treatment 1 showed similar values to treatments 2 to 4 and higher values than control. Since the most important parameter in color is redness (a* value) for cured meat products (Feng et al. 2016), these results indicated that the Chinese cabbage powder was very effective at increasing redness in the naturally cured sausages and showed the potential of this power for use as a replacement for nitrite. Treatments 1 and 2 showed similar (p>0.05) CIE b* values to the control (Table 2). However, treatments 3 and 4 exhibited higher (p<0.05) CIE b* values compared to the control and other treatments. These results were similar to those of Riel et al. (2017), who found that higher ingoing amounts of parsley extract powder resulted in higher b* values compared to traditionally cured sausages. Horsch et al. (2014) also reported that celery concentrate treatments were significantly more yellow (higher b* values) than the hams cured with nitrite and an increase of the concentration of celery concentrate lead to increased yellowness in the final ham products. Riel et al. (2017) and Horsch et al. (2014) indicated that the higher b* values might be related to the plant pigments in the vegetable concentrates. Therefore, our results suggested that when vegetable powder with increased nitrate contents was added to the meat products, the effect of the color of the vegetable powder could be minimized, and similar yellowness to that of the nitrite added products might be obtained in the alternatively cured sausages.

Residual nitrite, nitrosyl hemochrome, total pigments, and curing efficiency
The residual nitrite contents of the cooked pork sausages was 37.32, 14.68, 16.58, 22.24, and 16.35 ppm for the control, treatment 1, treatment 2, treatment 3, and treatment 4 (Table 3). Previous researchers have found this depletion of nitrite during product manufacturing. Xi et al. (2012) found that the amount of nitrite had reduced by about 75% of the initial concentration for frankfurters. Choi et al. (2020) also reported about 78% of the nitrite had depleted in ground pork products following the cooking and chilling process. In this study, the addition of a starter culture (S. carnosus) converted the nitrate from vegetable powder to nitrite in alternatively cured products although the added amount of starter culture varied depending on the treatments. Terns et al. (2011) indicated that the level of starter culture did not improve the cured meat properties in the indirectly cured, emulsified sausages. Nevertheless, all alternatively cured sausages had lower (p<0.05) residual nitrite contents than the control. Sebranek and Bacus (2007) found that the residual nitrite contents was lower in the naturally cured meat products than in the conventionally cured products. Similarly, Riel et al. (2017) reported that the residual nitrite contents of mortadella-type sausages produced with different levels of parsley extract powder were lower than the nitrite-added control. In this study, treatments 3 had higher (p<0.05) residual nitrite contents compared to other treatments.
Higher residual nitrite contents in treatment 3 among the alternatively cured products were expected because the highest ingoing amounts of nitrate (176 ppm) were from the Chinese cabbage powder in this study.
Nitrosyl hemochrome is a stable pink pigment formed by the reaction of nitric oxide converted from nitrite with myoglobin, resulting in a typical cured meat color after the cooking process (Cassens, 1997;Suman and Joseph, 2013). In this study, similar to the results of the CIE a* values, the nitrosyl hemochrome contents were higher (p<0.05) for all of the alternatively cured sausages than the control (Table 3). However, treatment 1 showed lower (p<0.05) nitrosyl hemochrome contents than treatments 2 to 4, which exhibited similar results (p>0.05). This finding suggested that the increase in redness of the alternatively cured sausages might be associated with an increase in the nitrosyl hemochrome, and sufficient amounts of nitrite were facilitated for the curing reactions, which resulted in the reduction of residual nitrite as shown in this study.
Similar to our results, Choi et al. (2020) found that the indirectly cured pork products with 0.2% or 0.4% white kimchi powder had a higher nitrosyl hemochrome contents than the conventionally cured control. Terns et al. (2011) also obtained similar results by adding celery juice powder and cherry powder to the indirectly cured, emulsified sausages. However, these results disagree with those of Sindelar et al. (2007b), who found no differences in the cured pigment contents between the nitrite-added control and the naturally cured hams treated with different concentrations of the celery juice powder and the incubation time. As a result, the results of the present study suggest that the addition of cabbage powder was very effective in the cured pigment formation in the alternatively cured products.
Total pigment contents of the products tested in this study ranged from 47.18 to 49.13 ppm and were lower than those found in previous research (Sindelar et al., 2007a) ranging from 58.1 to 92.5 ppm. However, compared to the control, the addition of Chinese cabbage powder or celery juice powder to the products increased (p<0.05) the total pigment contents.
However, these increases were similar (p>0.05) to each other. These results clearly followed the same trends as our CIE a* values. The relationship between nitrosyl hemochrome and total pigment contents was supported by Ahn and Maurer (1989), who speculated that less nitrosyl hemochrome formation could be the primary cause of a low total pigment. This has been previously confirmed by Sullivan et al. (2012), who reported that naturally cured frankfurters collected from commercial brands had the greatest amount of cured pigment and the highest total pigment contents. Thus, our results suggested that nitrite derived from vegetable sources by the starter culture was rapidly converted to nitric oxide, resulting in more nitrosyl hemochrome and total pigment contents in final products. In addition, it might be possible that the curing reaction in the alternatively cured products could occur faster because the vegetables such as the Chinese cabbage powder and celery juice powder possess inherent ascorbic acid that functions as a curing accelerator facilitated the conversion of nitrite to nitric oxide enhancing color development Sebranek et al., 2012;Zhu et al., 2018).
Curing efficiency of the pork products tested in this study ranged from 72.70% to 82.93% (Table 3). Cured pork sausages with Chinese cabbage powder or 0.4% celery juice powder had a higher (p<0.05) curing efficiency than the control with sodium nitrite. Furthermore, no differences (p>0.05) in curing efficiency were found between treatments 2 to 4. Kim et al. (2019b) reported that pork loins cured with fermented Swiss chard showed curing efficiency of 62.9% to 90.19% with the concentrations of fermented Swiss chard solutions, which was lower or higher than that of the control cured with 120 ppm nitrite (86.03%). Choi et al. (2020) found that the curing efficiency of indirectly cured meat products with white kimchi powder ranged from 75.26% to 79.34%, resulting in higher curing efficiency than the nitrite control. However, the lower nitrosyl hemochrome contents in treatment 1 resulted in decreasing curing efficiency and treatments 1 had less (p<0.05) curing efficiency than treatments 2 to 4. Therefore, these results suggested that the 0.15% addition (75 ppm ingoing nitrate) of Chinese cabbage powder did not show sufficient cured pigment and curing efficiency in the alternatively cured products even when it showed a higher curing efficiency than the control. Importantly, the addition of more than 0.25% Chinese cabbage powder (126 ppm ingoing nitrate) increased both the cured pigment and curing efficiency in the alternatively cured meat products.

Conclusion
Although cooking yield was slightly lower in the alternatively cured pork sausages with Chinese cabbage powder or celery juice powder, higher redness was found compared with the nitrite control. Moreover, less residual nitrite content were found in the alternatively cured meat products. In addition, the nitrite derived from the vegetable powder and starter culture with nitrate-reducing activity was more effectively involved in the curing reaction and more effective at the conversion of nitrite to nitric oxide, thereby resulting in higher cured pigment, total pigment, and curing efficiency. However, among the meat products with Chinese cabbage powder, 0.15% addition of Chinese cabbage powder (treatment 1) exhibited less cured pigment and curing efficiency, while the addition of more than 0.25% Chinese cabbage powder (126 ppm ingoing nitrate) positively affect the cured meat sausages with an increase in the cured pigment and curing efficiency comparable to those of the traditionally cured control. Therefore, this study indicated that Chinese cabbage powder showed efficacy as a new natural ingredient to replace synthetic nitrite for alternatively cured meat sausages. Further research is needed to explore the effectiveness of Chinese cabbage powder on the sensory attributes and shelf life of naturally cured meat products during storage for industrial practice.

Conflicts of Interest
The authors declare no potential conflicts of interest.