An Evaluation of Pig Type Regarding the Quality of Xuanwei Ham

: To determine the influence of pig type (Landrace, Wujin, or Tibetan fragrant) on the quality of Xuanwei ham, we measured the ham pH, color, fat content, and moisture content; used an E-nose (a device intended to detect odors or flavors); and analyzed flavoring substances using headspace solid-phase microextraction–gas chromatography, free amino acids using high-performance liquid chromatography, and microbial diversity using high-throughput sequencing. The ham types differed from each other in these attributes. The moisture and fat contents of Landrace pig ham were significantly lower than those of the other pig types, the brightness values of the Tibetan fragrant pig ham were significantly lower than those of the other pig types, and the redness values of the Landrace and Wujin pig hams were significantly higher than those of the Tibetan fragrant pig ham. The essential amino acid contents, e-wind odor response values, and volatile flavor substances of Wujin pig hams were significantly higher than those of the Tibetan fragrant pig ham, and the relative aldehyde contents of Wujin pig ham were significantly higher than those of the other pig types. The dominant microbial phyla in each ham type were assessed based on the species commonness, composition, and diversity and included taxa such as Actinobacteria and Ascomycetes and thick-walled bacteria such as Orphanomyces , Grass Spirochaetes , and Pseudoalteromonas . The microbial diversity and richness were the greatest in the Wujin pig ham. Of the three pigs, we conclude that the Wujin pig produces the best Xuanwei ham.


Introduction
Fermented meat products are made from fresh animal and poultry meats under certain natural conditions through fermentation by microorganisms or by enzymes.These meat products have unique flavors, a long preservation period, and certain health benefits.Many kinds of fermented meat products exist, among which fermented ham is one of the most popular (e.g., American country, Spanish Iberian, Italian Parma, and French Bayeux hams) [1,2].In China, Zhejiang Jinhua, Yunnan Xuanwei, and Jiangsu Rugao hams are all esteemed [2,3].
In 2009, in Yunnan Province, Xuanwei ham was listed as an intangible heritage cultural project; it is made from the pig's hind leg through a process involving trimming and shaping, salting and curing, and fermentation.Fermentation, the key to ham quality, is affected by various environmental factors and the raw pork materials themselves.The flavor quality of dry-cured hams processed at 35 • C under high pressure is lower than that at 0 and 20 • C [4].White spots and oil droplets on the surface of Spanish dry-cured hams appear at 52% humidity, while the ham quality at 78% and 52% humidity is similar, and there are fewer surface oil droplets at 85% humidity [5]; there was also a significant difference in 3-methylbutyraldehyde contents between Large White and Iberian porcine hams, with the higher concentration in the latter seeming to explain the higher consumer acceptance [6].Additionally, out of two crossbred ternary pigs (Yorkshire × Landrace × Duroc (YLD) and Yorkshire × Buckskin × Duroc (YBD)), YLD was better suited for dry-cured ham production [7].
Draft Xuanwei County Records [8] include a statement that "Xuanwei ham is famous all over the world, the climate makes it so".The climate of this monsoon region, a subtropical plateau, is unique and creates an environment suitable for fermenting Xuanwei ham.Throughout the Xuanwei region, the produced ham is collectively known as Xuanwei ham.Xuanwei area farmers breed a variety of pigs (Landrace, Duroc, Hampshire, York, Tibetan, and hybrids) [9].However, the use of different raw materials (pig types) to produce Xuanwei ham hampers industrialization and the standardization of products.
Aspects of Xuanwei ham (e.g., active ingredients, flavor, curing agents, and microorganisms) have been reported.Aqueous two-phase extraction was used to isolate, enrich, and protect antioxidant peptides in Xuanwei ham [10]; further, reasons for the bad flavor in this ham have been reported [11], as has the effect of using different proportions of KCl as a partial substitution for NaCl on volatile flavors [12].High-throughput sequencing has also been used to analyze the structure and diversity of Xuanwei ham microbial colonies [13].
Few studies have measured the effect of pig type on Xuanwei ham quality.There are many and varied types of ham combinations, and ham quality could be affected by production performance (pig litter weight), slaughtering (slaughtering rate and lean meat rate), meat quality (meat color, texture, pH, water loss, shear, cooked meat rate, and intramuscular fat), and organoleptic evaluation [14,15].In the past, research on the quality of different raw materials for Xuanwei ham was limited to the comparison of basic indicators, and there was a lack of deeper and comprehensive analysis.Regarding the previously mentioned issues, to apprise the effect of pig type (Landrace, Wujin, or Tibetan fragrant) on the quality of Xuanwei ham, we measured the ham pH, chroma, fat, and moisture contents and analyzed the volatile flavor substances, free amino acids, and microbial diversity, and the correlations between free amino acids, volatile flavor substances, and microorganisms are reported.The aim was to evaluate the most suitable raw material for processing Xuanwei ham and provide theoretical support for improving the quality of Xuanwei ham.

Materials and Reagents
On 8 October 2023, we purchased Xuanwei ham processed by Yunnan Yiji Food Co., Ltd.(Kunming, China), using Wujin, Landrace, and Tibetan fragrant pigs as the raw materials.These hams were processed by Yunnan Yiji Food Co., Ltd. on 10 August 2022, including raw material cutting, salt application, washing, and hanging.Fermentation began on 15 September 2022.The fermentation time was approximately 1 year.

Raw Material Handling
The same part of the bicep of each ham type (Wujin, Landrace, and Tibetan fragra pigs) was cut into pieces as shown in Figure 1, sealed in packages, and frozen. .W: Xuanwei ham processed using Wujin pig the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanw ham processed using Tibetan fragrant pig as the raw material.

pH
An acidimeter designed to determine solid acidity was used.Meat samples were c into 5 × 5 × 5 cm cubes parallel to the muscle fibers.A calibrated and cleaned acidimet probe was inserted directly into the center of each meat sample three times; the avera value was taken.

Moisture Content
Methods followed the National Standard of the People's Republic of China Nation Food Safety Standard [16].Three replicate measurements were taken, and the avera value was calculated.
We took a clean, flat weighing bo le made of glass, placed it in a drying oven at 1 °C~105 °C, heated it for 1.0 h, took it out, covered it, cooled it in a dryer for 0.5 h, weigh it, and repeated drying until the difference in mass between the two periods did not e ceed 2 mg, which is considered a constant weight.We weighed 2g~10g of the sample (a curate to 0.0001 g), placed it in this weighing bo le, weighed it accurately, and placed in a drying oven at 101 °C~105 °C.After drying for 2-4 h, we cooled it in a dryer for 0.5 before weighing.Then, we placed it in a drying oven at 101 °C~105 °C and dried for abo 1 h.We took it out and cooled it in a dryer for 0.5 h before weighing again.We repeat the above operation until the difference in mass between the two periods did not exce 2 mg, which is considered a constant weight.The moisture content in the sample w calculated using the following formula: .W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

pH
An acidimeter designed to determine solid acidity was used.Meat samples were cut into 5 × 5 × 5 cm cubes parallel to the muscle fibers.A calibrated and cleaned acidimeter probe was inserted directly into the center of each meat sample three times; the average value was taken.

Moisture Content
Methods followed the National Standard of the People's Republic of China National Food Safety Standard [16].Three replicate measurements were taken, and the average value was calculated.
We took a clean, flat weighing bottle made of glass, placed it in a drying oven at 101 • C~105 • C, heated it for 1.0 h, took it out, covered it, cooled it in a dryer for 0.5 h, weighed it, and repeated drying until the difference in mass between the two periods did not exceed 2 mg, which is considered a constant weight.We weighed 2g~10g of the sample (accurate to 0.0001 g), placed it in this weighing bottle, weighed it accurately, and placed it in a drying oven at 101 • C~105 • C.After drying for 2-4 h, we cooled it in a dryer for 0.5 h before weighing.Then, we placed it in a drying oven at 101 • C~105 • C and dried for about 1 h.We took it out and cooled it in a dryer for 0.5 h before weighing again.We repeated the above operation until the difference in mass between the two periods did not exceed 2 mg, which is considered a constant weight.The moisture content in the sample was calculated using the following formula: In the formula, X-moisture content in the sample (unit: g/100 g); m1-the mass of the weighing bottle (with sea sand and glass rod) and the sample, in grams; m2-the mass of the weighing bottle (with sea sand and glass rod) and the dried sample, in grams; m3-mass of weighing bottle (with sea sand and glass rod), in grams; 100-unit conversion factor.

Fat Content
Methods followed the National Standard of the People's Republic of China National Food Safety Standard [17].Three replicate measurements were taken, and the average value was calculated.
For sample processing, we weighed 2 g~5 g of the sample, accurate to 0.001 g, and transferred it to a filter paper cylinder.
For extraction, we placed a filter paper cylinder in the extraction cylinder of a Soxhlet extractor, connected the receiving bottle that was dried to a constant weight, and added anhydrous ether or petroleum ether to two-thirds of the volume of the bottle at the upper end of the condenser of the extractor.We then heated it in a water bath to continuously reflux and extract anhydrous ether or petroleum ether.Generally, extraction takes 6-10 h.At the end of extraction, we used a frosted glass rod to take 1 drop of extraction solution.If there were no oil spots on the frosted glass rod, it indicated that the extraction was complete.
For weighing, we removed the receiving bottle, recovered anhydrous ether or petroleum ether, and waited for 1 mL~2 mL of solvent to remain in the receiving bottle.We then steam-dried it in a water bath, dried it at 100 • C ± 5 • C for 1 h, cooled it in a dryer for 0.5 h, and weighed.We repeated the above steps until the weight was constant (until the difference between the two weights did not exceed 2 mg).The fat content in the sample was calculated using the following formula: In the formula, X-fat content in the sample (unit: g/100 g); m1-the content of the receiving bottle and fat at constant weight, in grams; m0-mass of the receiving bottle, in grams; m2-mass of the sample, in grams; 100-unit conversion factor.

Color
A colorimeter designed to determine sample colors was used.A lean part of the ham was selected and cut into 5 mm slices.At three random points, the color was measured three times (sample brightness (L*), redness (a*), and yellowness (b*)).

Free Amino Acids
A 5 g tissue sample was crushed and placed in a 10 mL centrifuge tube, to which 5 mL of 0.01 mol/L hydrochloric acid was added.The sample was mixed, placed in a boiling water bath for 30 min, and centrifuged at 10,000 rpm for 10 min.The supernatant was collected, and the precipitate was added to 4 mL of 0.01 mol/L hydrochloric acid suspension, exposed to ultrasound for 5 min, and centrifuged.The supernatant was combined, and the sample was fixed to 10 mL.
For on-line pre-column derivatization, using Agilent's automated on-line derivatization method, primary amino acids with OPA and secondary amino acids with FMOC were derived and passed through a column.
The chromatographic conditions were as follows: ZORBAX Eclipse AAA (4.6 × 150 mm, 3.5 µm); detection signal: UV 338 nm (0-19 min) and 266 nm (19.01-25 min); mobile phase A: 40 mM sodium dihydrogen phosphate (pH 7.8); mobile phase B: acetonitrile/methanol/water = 45/45/10; flow rate: 1.0 mL/min; column temperature: 45 • C. The elution gradient is presented in Table 1.For substance detection, the sample was stirred, and 10 mL was placed in a 20 mL headspace vial and equilibrated at 40 • C for 30 min, followed by immediate on-line detection.The detection parameters involved a cleaning time of 120 s and a detection time of 120 s.The sample was then washed for 30 min at 40 • C. The substances responded by each sensor of the electronic nose are shown in Table 2. Flavoring substances were extracted and detected using headspace solid-phase microextraction (SPME)-GC-MS.Extraction conditions involved a 5 g sample added to a 20 mL extraction vial, to which 100 µL of 2,4,6-trimethylpyridine (0.05 mg/mL) was added as an internal standard.The vial was sealed and placed in a water bath at 85 • C, with a magnetic stirring speed of 500 rpm.The sample was equilibrated for 20 min, and then the needle was inserted into the headspace of the vial to extract volatiles for 30 min.The needle was activated in the gas inlet port for 20 min at 250 • C before use.The GC conditions were as follows: inlet temperature, 250 • C; gas interface temperature, 250 • C; carrier gas flow rate, 1.5 mL/min; split ratio, 4:1.The heating procedure was as follows: initially 40 • C, maintained for 5 min, and then increased by 5 • C/min to 250 • C and maintained for 10 min.The MS conditions were as follows: ion source temperature, 230 • C; quadruple rod temperature, 150 • C; EI ionization, 70 eV; full scan, 35-550 da.

Microbiological Detection
Wekemo Tech Group Co., Ltd.(Shenzhen, China) was commissioned to perform high-throughput sequencing and microbial DNA extraction from the ham samples.PCR amplification and sequencing methods followed the manufacturer's instructions; the Illumina NovaSeq platform was used to bipartite sequence the sequenced samples.

Data Processing
Data were collated using Microsoft Excel 2019 and analyzed via ANOVA using IBM SPSS Statistics 26; values are expressed as means ± standard deviations (n = 3) with a significance level of p < 0.05.Correlation analysis of microbial diversity with amino acids and flavoring substances was performed using the Wekemo Bioincloud platform.

Physicochemical Comparison of Raw Hams
The moisture and fat contents of the Wujin and Tibetan fragrant pig hams were significantly higher than those of the Landrace pig ham (p < 0.05); the fat and moisture contents of the Wujin pig ham (8.80 g/100 g and 49.27 g/100 g, respectively) were the highest, and the moisture content and pH of the Landrace pig ham (29.39 g/100 g and 5.74, respectively) were the lowest (Table 3).The higher the intramuscular fat content is, the higher the muscle water-holding capacity is, which is related to muscle pH (the higher the pH is, the lower the water loss is) [18].The large standard deviation for the moisture content in the Landrace pig ham may be related to the environment in which the samples were stored prior to analysis and the increased frequency of exposure to air during weighing, resulting in greater intervals between analyses of parallel samples.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.Different lowercase letters indicate significant differences between varieties (p < 0.05).
There was no significant difference in L* values between the Wujin and Landrace pig hams (p < 0.05), but the L* values of both were significantly higher than those of the Tibetan fragrant pig ham (p < 0.05).The a* and b* values differed significantly between hams, with Tibetan fragrant pig ham presenting values significantly lower than those for either other pig ham (p < 0.05).This indicates that the color of Tibetan fragrant pig ham is less bright than that of either other pig type, probably because the myoglobin content of Wujin and Landrace pigs is greater.The main chromogenic substances in ham are myoglobin-like and hemoglobin [11], and the content and chemical state of myoglobin determine muscle color (the higher the content is, the brighter the meat is) [19].
Yang et al. [20] verified the correlation between meat color and myoglobin by investigating the differences in meat color and myoglobin content between Rongchang pig, Yorkshire pig, Rongchang pig (Yuerong pig), Duroc pig, Landrace pig, and Landrace pig (Duroc three-yuan pig).They concluded that there is a significant positive correlation between pork a* value, total myoglobin content, and oxygenated myoglobin content.Liu Q et al. [21] investigated the correlation of myoglobin content with meat color and other traits in eight hybrid heterogeneous pork populations.In the phenotypic association between myoglobin content and meat color, there was a moderate to high correlation.

Free Amino Acids in Raw Hams
Free amino acids can be fresh (e.g., Asp and Glu), sweet (e.g., Thr, Ala, Ser, Gly, and Pro), or bitter (His, Val, Met, Lys, Trp, Phe, Ile, Leu, Arg, and Tyr) according to their flavor characteristics.During ham fermentation and maturation, amino acids are mainly produced via peptide and protein hydrolysis under the action of enzymes, and the quantities of different amino acids determine the flavor of dry-cured ham [22].
The TAV (taste activity value) is the ratio of an amino acid's content to its threshold (calculation formula: TAV = C (content of amino acid)/T (threshold)).When TAV > 1, it indicates that the substance significantly contributes to flavor formation.The TAVs of Asp, Glu, Ala, Ser, His, Gly, Val, Met, and Lys in the Wujin and Landrace pig hams were >1; of these, the TAV of Glu was much greater, contributing to the ham's fresh flavor.The TAVs of Glu, Ala, Val, Met, and Lys in the Tibetan fragrant pig ham were >1, with the TAV of Glu being much greater, also contributing more to the fresh flavor of this ham (Table 4).In terms of the total amino acid (TAA) content, there was no significant difference between the Wujin and Landrace pig hams (p < 0.05).The TAA content of the Wujin and Landrace pig hams was approximately twice that of the Tibetan fragrant pig ham; therefore, the overall flavor of the Wujin and Landrace pig hams was stronger than that of the Tibetan fragrant pig ham.
Lys and Leu were the most abundant essential amino acids (EAAs) in each ham type, and their contents in the Wujin pig ham were significantly higher than those in either other type (p < 0.05).In addition to participating in protein synthesis, Lys and Leu promote enzyme and antibody production and improve immune system functioning [23].Asp, Glu, and Arg are the most abundant non-essential amino acids, but their contents in the Tibetan fragrant pig ham were significantly lower than those in either other type (p < 0.05).Arg plays an important role in vascular homeostasis, spermatogenesis, and infant growth and is considered an EAA, especially if a child's growth requires a high degree of metabolism and endogenous synthesis cannot meet metabolic demand [23].W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.Different lowercase letters indicate significant differences between different varieties (p < 0.05).

Electronic Nose for Raw Hams
The sensor response values for the odor composition for each ham type (Figure 2, Left) revealed little difference between the Landrace and Tibetan fragrant pig hams compared with the Wujin pig ham.The values for the Wujin pig ham from the S1, S2, S4-8, and S10 sensors were significantly stronger than those for the other ham types.This indicates that Wujin pig ham has higher alcohol, aldehyde, short-chain alkane, sulfide, organic amine, and aromatic compound contents than the other ham types, and the overall odor is more prominent.
The sensor response values for the odor composition for each ham type (Figure 2, Left) revealed li le difference between the Landrace and Tibetan fragrant pig hams compared with the Wujin pig ham.The values for the Wujin pig ham from the S1, S2, S4-8, and S10 sensors were significantly stronger than those for the other ham types.This indicates that Wujin pig ham has higher alcohol, aldehyde, short-chain alkane, sulfide, organic amine, and aromatic compound contents than the other ham types, and the overall odor is more prominent.
The contribution rates of the first and second principal components were 88.8567% and 9.6626%, respectively (their sum is 98.5193%) (Figure 2, Right).In the principal component analysis, the greater the relative distance between samples is, the greater the difference in their odor is.A clear distinction exists between the three ham types, indicating clear differences in their odor characteristics (Figure 2, Right).
Figure 2. Radar plots (Left) and PCA analyses (Right) of an electronic nose responding to substances in different raw ham varieties.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Volatile Flavor Substances of Raw Hams
A total of 59 volatile flavor compounds (aldehydes, esters, alcohols, hydrocarbons, and ketones) were detected in the three ham types, of which 29 were common in all hams (Table 5).The OAV (odor activity value) reveals the contribution of aroma components to The contribution rates of the first and second principal components were 88.8567% and 9.6626%, respectively (their sum is 98.5193%) (Figure 2, Right).In the principal component analysis, the greater the relative distance between samples is, the greater the difference in their odor is.A clear distinction exists between the three ham types, indicating clear differences in their odor characteristics (Figure 2, Right).

Volatile Flavor Substances of Raw Hams
A total of 59 volatile flavor compounds (aldehydes, esters, alcohols, hydrocarbons, and ketones) were detected in the three ham types, of which 29 were common in all hams (Table 5).The OAV (odor activity value) reveals the contribution of aroma components to the food aroma system in two dimensions: concentration and threshold.Its calculation formula is OAV = ρ (volatile flavor content)/T (threshold).In total, 45 volatile flavor compounds were detected in the Wujin pig ham, totaling 66.56 µg/g, of which 9 had an OAV of >1; 49 volatile flavor compounds were detected in the Landrace pig ham, totaling 47.75 µg/g, of which 11 had an OAV of >1; and 39 volatile flavor compounds were detected in the Tibetan fragrant pig ham, totaling 28.20 µg/g, of which 8 had an OAV of >1.
Aldehydes mainly originate from the oxidative degradation of lipids and partly from protein hydrolysis and amino acid degradation [25].They have fruity and fatty aromas [26].Nonanal and hexadecanal were the main aldehydes in each ham type; the OAV of nonanal was much greater than one and contributed most to the fatty aroma of each ham.The almond-like flavor of benzaldehyde is derived from amino acids formed via the Strecker reaction [27], and the benzaldehyde content of the Wujin pig ham was significantly higher than that in either other ham type, indicating that its amino acid content was significantly higher, consistent with the results in Section 3.2.As the threshold value of aldehydes is low, small amounts can produce a strong, fresh aroma.The aldehyde content of the Wujin pig ham was significantly higher than that of either other ham type, which is consistent with the electronic nose test results, indicating that the fat aroma of Wujin pig ham is more intense.
Many methyl and ethyl esters were detected in each ham type.Esters are derived from the esterification of carboxylic acids and alcohols; the esters of short-chain acids have a fruity and sweet flavor, while long-chain acids produce a fatty odor [28].The Wujin pig ham had significantly higher ester levels than either other ham type, suggesting that it produces more ethanol during microbial fermentation [29].
Alcohols were detected in fewer types and lower amounts in each ham type, with most having a high threshold and not contributing significantly to the meat product flavor [30].1-Octen-3-ol was detected in each ham type and originates from the β-oxidation of unsaturated fatty acids; it has a strong mushroom flavor [31].The 1-Octen-3-ol content in the Wujin pig ham was significantly higher than in the other types, possibly because of its higher unsaturated fatty acid content.
Alkanes are important components of volatile flavor substances, mainly derived from lipid oxidation.As their thresholds are usually very high and their odor is usually weak or even non-existent, their influence on ham flavor is not significant [30].
Ketones are produced by lipid oxidation and are usually associated with fruity, pungent, and fatty flavors [26].The only ketone detected, 2-pentadecanone, was present in the Wujin pig ham and may be a key compound in forming its distinctive flavor.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.Different lowercase letters indicate significant differences between different varieties (p < 0.05).

Alpha Diversity
Chao1 is commonly used to indicate the total number of microorganism species, while the Shannon and Simpson indexes estimate the microbial diversity in samples, with higher values indicating higher species diversity.The alpha diversity values of the Wujin pig ham were the highest at 550.360 (Chao1) and 0.912 (Simpson's index), indicating greater microbial community richness and diversity.The alpha diversity values of the Landrace pig ham were the lowest at 208.595 (Chao1) and 0.809 (Simpson's index), indicating lower microbial community richness and diversity (Table 6).W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Shared Species
In petal diagrams, the number of species endemic to a subgroup is in the petals, and the number of species common to all subgroups is in the center (Figure 3).An OTU (Operational Taxonomic Unit), also known as a taxonomic unit, is a set of identical markers (strain, species, genus, etc.) artificially assigned for analysis in phylogenetic or population genetics research.In a microbial diversity analysis, all sequences are divided into OTUs based on different similarity levels.Generally, if the similarity between sequences is higher than 97% (species level), it can be defined as an OTU, and each OTU represents a species.The higher the number of OTUs is, the higher the microbial abundance is.In total, there were 250 OTUs common to all three ham types: 604 (Wujin pig), 572 (Tibetan fragrant pig), and 316 (Landrace pig).The Chao1 index reflects the number of OTUs, and the two correlate positively; the greater the Chao1 index is, the greater the number of OTUs is and the greater the species richness is.The number of OTUs was highest in Wujin pig, consistent with the α-diversity results, further demonstrating that the microbial community richness and diversity in hams from this pig were the highest of all three types.there were 250 OTUs common to all three ham types: 604 (Wujin pig), 572 (Tibetan fragrant pig), and 316 (Landrace pig).The Chao1 index reflects the number of OTUs, and the two correlate positively; the greater the Chao1 index is, the greater the number of OTUs is and the greater the species richness is.The number of OTUs was highest in Wujin pig, consistent with the α-diversity results, further demonstrating that the microbial community richness and diversity in hams from this pig were the highest of all three types.Figure 3. Wayne diagram of microbial communities of hams from different raw materials.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Species Composition
Relative Abundance of Phyla and Genera Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Species Composition Relative Abundance of Phyla and Genera
The distribution of the top 20 microbial colonies for each ham type is depicted in Figure 4 in terms of relative abundance at the phylum and genus levels.The distribution of the top 20 microbial colonies for each ham type is depicted in Figure 4 in terms of relative abundance at the phylum and genus levels.
The dominant phyla in each ham type included Pseudomonadota, Ascomycota, Artverviricota, Actinomycetota, and Bacillota.For each ham type, Pseudomonadota was the most abundant taxon; its relative abundance was significantly higher in the Wujin and Tibetan fragrant pig hams than in the Landrace pig ham.The relative abundances of Ascomycota and Artverviricota in the Landrace pig ham were significantly higher than in the other ham types.
The dominant genera in each ham type included Sarocladium, Vibrio, Gammaretrovirus, Herbaspirillum, Klebsiella, and Pseudoalteromonas.The relative abundances of Sarocladium, Gammaretrovirus, and Herbaspirillum in the Landrace pig ham were significantly higher than in either other type; the relative abundance of Vibrio and Pseudoalteromonas in the Wujin pig ham was significantly higher than in either other type; and the relative abundance of Klebsiella in the Tibetan fragrant pig ham was significantly higher than in either other type.and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Heat Map of Species Abundance Clustering
In the Wujin pig ham groups (W1, W2, and W3), Landrace pig ham groups (P1, P2, and P3), and Tibetan fragrant pig ham groups (Z1, Z2, and Z3), different colors represent different abundances.The redder the color is, the higher the abundance is, and the bluer the color is, the lower the abundance is.
The microorganisms in each ham type differed significantly in their phylum and genus composition (Figure 5).At the phylum level, Basidiomycota from P3; Chrysiogenota from W2; Bacillota, Cyanobacteriota, and Deinococcota from W3; Mucoromycota from Z1; Verrucomicrobiota, Bacteroidota, Thermoproteota, and Thermodesulfobacteriota from Z2; and Campylobacterota from Z3 had the strongest red color, indicating that these hams had the highest relative microorganism abundance.There was only one dominant phylum in the Landrace pig ham group, four dominant phyla in the Wujin pig ham group, and six dominant phyla in the Tibetan fragrant pig ham group, indicating that Tibetan fragrant and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.
The dominant phyla in each ham type included Pseudomonadota, Ascomycota, Artverviricota, Actinomycetota, and Bacillota.For each ham type, Pseudomonadota was the most abundant taxon; its relative abundance was significantly higher in the Wujin and Tibetan fragrant pig hams than in the Landrace pig ham.The relative abundances of Ascomycota and Artverviricota in the Landrace pig ham were significantly higher than in the other ham types.
The dominant genera in each ham type included Sarocladium, Vibrio, Gammaretrovirus, Herbaspirillum, Klebsiella, and Pseudoalteromonas.The relative abundances of Saro-cladium, Gammaretrovirus, and Herbaspirillum in the Landrace pig ham were significantly higher than in either other type; the relative abundance of Vibrio and Pseudoalteromonas in the Wujin pig ham was significantly higher than in either other type; and the relative abundance of Klebsiella in the Tibetan fragrant pig ham was significantly higher than in either other type.

Heat Map of Species Abundance Clustering
In the Wujin pig ham groups (W1, W2, and W3), Landrace pig ham groups (P1, P2, and P3), and Tibetan fragrant pig ham groups (Z1, Z2, and Z3), different colors represent different abundances.The redder the color is, the higher the abundance is, and the bluer the color is, the lower the abundance is.
The microorganisms in each ham type differed significantly in their phylum and genus composition (Figure 5).At the phylum level, Basidiomycota from P3; Chrysiogenota from W2; Bacillota, Cyanobacteriota, and Deinococcota from W3; Mucoromycota from Z1; Verrucomicrobiota, Bacteroidota, Thermoproteota, and Thermodesulfobacteriota from Z2; and Campylobacterota from Z3 had the strongest red color, indicating that these hams had the highest relative microorganism abundance.There was only one dominant phylum in the Landrace pig ham group, four dominant phyla in the Wujin pig ham group, and six dominant phyla in the Tibetan fragrant pig ham group, indicating that Tibetan fragrant pigs have the largest microbial community at the phylum level.At the genus level, Malassezia and Lasiodiplodia from P3, Microbacterium from W2, Salmonella and Ralstonia from W3, Saccharomyces from Z1, and Klebsiella and Pseudomonas from Z2 had the strongest red color, indicating that the relative abundance of microorganisms in these hams was the highest.There were two dominant genera in the Landrace pig ham group, three in the Wujin pig ham group, and three in the Tibetan fragrant pig ham group.Compared with the Wujin and Tibetan fragrant pig ham groups, the Landrace pig ham group had the smallest microbial community at both the phylum and genus levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Correlations between Free Amino Acids and Microorganisms
Based on their relative abundances, the top 30 genera were selected for correlation analysis with major flavor-presenting amino acids with a TAV of >1.A total of 88 flavorpresenting amino acids correlated with microorganisms, of which 19 were positively correlated and 69 were negatively correlated (Figure 6).The relative abundances of Die ia and Pseudoclavibacter showed a highly significant positive correlation with Ala content (p < 0.01); the relative abundance of Bacillus was significantly positively correlated with His content (p < 0.05); and the relative abundances of Synechococcus, Varicellovirus, Chromobacterium, Alicycliphilus, Croceicoccus, Nitrospira, Solibacillus, and Microcystis were significantly positively correlated with Val and Asp contents (p < 0.05).Among the genera that correlated positively with flavoring amino acids, 91% were bacteria and 9% belonged to Heunggongvirae, indicating that fermentation via bacteria is closely related to ham

. Correlations between Free Amino Acids and Microorganisms
Based on their relative abundances, the top 30 genera were selected for correlation analysis with major flavor-presenting amino acids with a TAV of >1.A total of 88 flavorpresenting amino acids correlated with microorganisms, of which 19 were positively correlated and 69 were negatively correlated (Figure 6).The relative abundances of Dietzia and Pseudoclavibacter showed a highly significant positive correlation with Ala content (p < 0.01); the relative abundance of Bacillus was significantly positively correlated with His content (p < 0.05); and the relative abundances of Synechococcus, Varicellovirus, Chromobacterium, Alicycliphilus, Croceicoccus, Nitrospira, Solibacillus, and Microcystis were significantly positively correlated with Val and Asp contents (p < 0.05).Among the genera that correlated positively with flavoring amino acids, 91% were bacteria and 9% belonged to Heunggongvirae, indicating that fermentation via bacteria is closely related to ham freshness, sweetness, and bitterness and plays an important role in ham flavoring.

Correlations between Volatile Flavor Substances and Microorganisms
The top 30 genera ranked in terms of relative abundance were selected for correl analysis with major volatile flavor compounds with OAVs of >1.A total of 128 flavor pounds correlated with microorganisms, of which 43 were positively correlated an were negatively correlated (Figure 7).The relative abundance of Gammaretrovirus c lated significantly and positively with 2-pentylfuran, n-hexanal, methyl octanoate, a octanal contents (p < 0.05); the relative abundance of Variovorax correlated signific and positively with 2-pentylfuran, n-hexanal, and methyl octanoate contents (p < 0.05 relative abundance of Finegoldia correlated significantly and positively with zeneacetaldehyde and benzaldehyde contents (p < 0.05); Synechococcus, Varicellov Chromobacterium, Alicycliphilus, Croceicoccus, Nitrospira, Solibacillus, and Microc correlated significantly and positively with the relative abundances of methyl isoval and butanal,3-methyl-and 2,6-dimethylpyrazine (p < 0.05); Elizabethkingia correlate nificantly and positively with the relative abundances of benzeneacetaldehyde and zaldehyde (p < 0.05) and highly significantly and positively with 2,6-dimethylpyr

Correlations between Volatile Flavor Substances and Microorganisms
The top 30 genera ranked in terms of relative abundance were selected for correlation analysis with major volatile flavor compounds with OAVs of >1.A total of 128 flavor compounds correlated with microorganisms, of which 43 were positively correlated and 85 were negatively correlated (Figure 7).The relative abundance of Gammaretrovirus correlated significantly and positively with 2-pentylfuran, n-hexanal, methyl octanoate, and n-octanal contents (p < 0.05); the relative abundance of Variovorax correlated significantly and positively with 2-pentylfuran, n-hexanal, and methyl octanoate contents (p < 0.05); the relative abundance of Finegoldia correlated significantly and positively with benzeneacetaldehyde and benzaldehyde contents (p < 0.05); Synechococcus, Varicellovirus, Chromobacterium, Alicycliphilus, Croceicoccus, Nitrospira, Solibacillus, and Microcystis corre-lated significantly and positively with the relative abundances of methyl isovalerate and butanal,3-methyl-and 2,6-dimethylpyrazine (p < 0.05); Elizabethkingia correlated significantly and positively with the relative abundances of benzeneacetaldehyde and benzaldehyde (p < 0.05) and highly significantly and positively with 2,6-dimethylpyrazine contents (p < 0.01); the relative abundance of Salmonella was significantly and positively correlated with benzeneacetaldehyde, benzaldehyde, and 2,6-dimethylpyrazine contents (p < 0.05); the relative abundance of Dietzia was highly and significantly positively correlated with benzeneacetaldehyde and benzaldehyde contents (p < 0.05); and the relative abundance of Ralstonia correlated significantly and positively with benzeneacetaldehyde and benzaldehyde contents (p < 0.05).Of the genera that correlated positively with flavor compounds, 87% were bacteria, 6.5% belonged to Heunggongvirae, and 6.5% belonged to Pararnavirae, similar to the correlation results for flavor-presenting amino acids.Thus, bacteria also play an important role in forming flavor compounds.(p < 0.05); the relative abundance of Die ia was highly and significantly positively correlated with benzeneacetaldehyde and benzaldehyde contents (p < 0.05); and the relative abundance of Ralstonia correlated significantly and positively with benzeneacetaldehyde and benzaldehyde contents (p < 0.05).Of the genera that correlated positively with flavor compounds, 87% were bacteria, 6.5% belonged to Heunggongvirae, and 6.5% belonged to Pararnavirae, similar to the correlation results for flavor-presenting amino acids.Thus, bacteria also play an important role in forming flavor compounds.

Discussion
Zhejiang Jinhua, Yunnan Xuanwei, and Jiangsu Rugao hams are coveted by consumers in China.Zhejiang Jinhua ham is made from the hind leg of Jinhua "two-headed crow", and Jiangsu Rugao ham is made from the fresh leg of "East String Pig".However, the lack of standardization in raw meat sources for Yunnan Xuanwei ham has hindered the commercialization of this product.We selected hams processed from three different raw materials (Wujin, Landrace, and Tibetan fragrant pigs) from the Xuanwei area.The physicochemical properties, free amino acids, volatile flavoring substances, and microbial communities of the hams made using each of these pig types were measured, and their free amino acids, volatile flavoring substances, and microorganisms were compared.
A pH between 6.0 and 6.2 increases microbiological risk, whereas one between 5.6

Discussion
Zhejiang Jinhua, Yunnan Xuanwei, and Jiangsu Rugao hams are coveted by consumers in China.Zhejiang Jinhua ham is made from the hind leg of Jinhua "two-headed crow", and Jiangsu Rugao ham is made from the fresh leg of "East String Pig".However, the lack of standardization in raw meat sources for Yunnan Xuanwei ham has hindered the commercialization of this product.We selected hams processed from three different raw materials (Wujin, Landrace, and Tibetan fragrant pigs) from the Xuanwei area.The physicochemical properties, free amino acids, volatile flavoring substances, and microbial communities of the hams made using each of these pig types were measured, and their free amino acids, volatile flavoring substances, and microorganisms were compared.
A pH between 6.0 and 6.2 increases microbiological risk, whereas one between 5.6 and 6.0 produces more desirable saltiness, color, and texture [34].The ham pH values ranged from 5.74 to 5.89.Accordingly, the pH of each ham made from each pig type was acceptable and positively affected ham quality.A higher pH may lead to a shorter shelf life, but it may also in a higher water-holding capacity [35].This is consistent with our detection of the highest pH and moisture content in the Wujin pig ham.The a* values of the Wujin and Landrace pig hams were higher than those of the Tibetan fragrant pig ham, indicating that the meat color of the former two was redder than the latter.Myoglobin and hemoglobin are the main components of ham color, and a lack of fat protection may allow more myoglobin or hemoglobin to be oxidized during curing, producing darker-colored meat [36].Therefore, fat also has an impact on the color of ham.The fat content of the Landrace pig ham was lower than that of the other two types, with its a* value being the highest among the three and L* being lower than that in the Wujin pig ham, showing a dark meat color characteristic.Wujin pig ham has the highest fat content, which can reduce the oxidation of myoglobin and hemoglobin during fermentation and prevent the darkening of the meat color.From the perspective of physical and chemical properties, Wujin pig ham has the best overall performance.
Ham fermentation is mainly based on the interactions of microbial communities.The various enzymes secreted by microorganisms play an important role in generating flavor compounds in a series of complex chemical reactions.
The richness and diversity of microbial communities in the samples processed from Wujin pig were the highest among the three ham types.Pseudomonadota is a cold-feeding aerobic spoilage bacterium phylum that is typically "sticky" and indicates "spoilage".Its presence can reflect the hygienic status of a product [37].Pseudomonadota was the most abundant phylum in each ham type and can promote amino acid production through deamination and decarboxylation reactions, enriching the flavor characteristics of ham.The large amount of Pseudomonadota in the Wujin pig ham supported the EAA results, further indicating that the changes in EAAs were influenced by the pig type and that changes in EAAs are influenced by microbial communities [38].
Among the flavoring amino acids that correlated positively with genera, those in the Wujin pig ham were higher in number than those in the other ham types, possibly because there was greater protease activity or peptidase in Wujin pig.Genera promote protein hydrolysis, influencing free amino acid composition and content.The higher the total free amino acid content is, the higher the nutritional value is [39].The free amino acid contents of the Wujin pig ham were higher than those of the other types, showing more taste characteristics and nutritional value.
Generating ham flavor compounds is inseparable from a series of reactions, including protein hydrolysis, lipid oxidation, Maillard reaction, and microbial active enzymes.In total, 87% of flora that correlated positively with flavor compounds were bacteria, indicating that bacteria can better decompose lipids and proteins and produce flavor compound precursors while meeting their own nutritional needs [40].Aldehydes are the most important contributors to ham flavor and occurred in the highest amounts in the Wujin pig ham.Their flavor compounds were the most abundant in the ham types, suggesting that the overall flavor of the Wujin pig ham was better than that of the other two.

Conclusions
We analyzed the physicochemical properties, free amino acids, volatile flavor compounds, and microorganisms of Xuanwei hams processed from three kinds of raw materials (Wujin, Landrace, and Tibetan fragrant pigs).The results showed no significant difference in pH among the Xuanwei pig ham types, and they were all within the normal range.
Compared with the Landrace pig ham, the Wujin pig ham and Tibetan fragrant pig ham had higher moisture and fat contents.The a* values, free amino acid contents, and volatile flavor substance contents of the Wujin pig ham and Landrace pig ham were higher than those of the Tibetan fragrant pig ham.There was no significant difference in Simpson values among the pig types.Compared with the Landrace pig ham, the Chao1 and Shannon values of the Wujin and Tibetan fragrant pig hams were higher.Their microbial diversity was also higher.
The data show that the Wujin pig ham had the best physicochemical characteristics, including the highest EAA content and total amino acids (which have high nutritional value) and the highest aldehyde and volatile flavoring compound contents of the three types.The microbial diversity and abundance in the Wujin pig ham also positively affected fermentation.Accordingly, we conclude that the flavor of Xuanwei ham is the best when produced with Wujin pig as the raw material.Using this pig would improve the nutritional value of Xuanwei ham and its commercial value as a standardized product.

( a )Figure 1 .
Figure1.Schematic diagram of sampling the biceps femoris muscle of Xuanwei ham from differe pig types (Wujin, Landrace, and Tibetan fragrant).W: Xuanwei ham processed using Wujin pig the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanw ham processed using Tibetan fragrant pig as the raw material.

Figure 1 .
Figure 1.Schematic diagram of sampling the biceps femoris muscle of Xuanwei ham from different pig types (Wujin, Landrace, and Tibetan fragrant).W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 2 .
Figure 2.Radar plots (Left) and PCA analyses (Right) of an electronic nose responding to substances in different raw ham varieties.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 3 .
Figure 3. Wayne diagram of microbial communities of hams from different raw materials.W:Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 4 .
Figure 4. Distribution of microbial communities of different varieties of raw hams at phylum (Left)and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 4 .
Figure 4. Distribution of microbial communities of different varieties of raw hams at phylum (Left)and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Fermentation 2024 ,
10, x FOR PEER REVIEW 13 of 19pigs have the largest microbial community at the phylum level.At the genus level, Malassezia and Lasiodiplodia from P3, Microbacterium from W2, Salmonella and Ralstonia from W3, Saccharomyces from Z1, and Klebsiella and Pseudomonas from Z2 had the strongest red color, indicating that the relative abundance of microorganisms in these hams was the highest.There were two dominant genera in the Landrace pig ham group, three in the Wujin pig ham group, and three in the Tibetan fragrant pig ham group.Compared with the Wujin and Tibetan fragrant pig ham groups, the Landrace pig ham group had the smallest microbial community at both the phylum and genus levels.

Figure 5 .
Figure 5. Heat map distribution results for different varieties of raw hams at the phylum (Left) and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 5 .
Figure 5. Heat map distribution results for different varieties of raw hams at the phylum (Left) and genus (Right) levels.W: Xuanwei ham processed using Wujin pig as the raw material; P: Xuanwei ham processed using Landrace pig as the raw material; Z: Xuanwei ham processed using Tibetan fragrant pig as the raw material.

Figure 7 .
Figure 7. Heat map of correlations between major volatile flavor substances and microorganisms.* Significant at p < 0.05; ** significant at p < 0.01.

Figure 7 .
Figure 7. Heat map of correlations between major volatile flavor substances and microorganisms.* Significant at p < 0.05; ** significant at p < 0.01.

Table 2 .
Substances responsive to the electronic nose sensor.

Table 3 .
Comparison of physicochemical indexes of different raw ham varieties.

Table 4 .
Free amino acid content and TAVs of different raw ham varieties.

Table 5 .
Volatile flavor content and OAVs of different raw ham varieties.

Table 6 .
The α-diversity of microorganisms in different raw ham varieties.