Inclusion of Saccharomyces cerevisiae var. boulardii RC009 and Pediococcus pentosaceus RC007 as a Probiotic Additive in Pigs’ Postweaning Diets and Its Effect on Meat Composition, Carcass Characteristics, and Fatty Acids Profile after Slaughter

Te postweaning is recognized as one of the most challenging stages of pig growth that can afect their lifetime productivity. Te aim of the study was to evaluate the inclusion of Saccharomyces cerevisiae var. boulardii RC009 and Pediococcus pentosaceus RC007 as a probiotic additive in pigs’ postweaning diets and its efect on meat composition, carcass characteristics, and fatty acids profle after slaughter. Te following three treatments (550 animals each) were included: T1: control balanced diet (CD), T2: CD with S. boulardii RC009 (1 × 109CFU/kg feed), and T3: CD with P. pentosaceus RC007 (1 × 109CFU/kg feed). Te additive was administered throughout the postweaning phase (49 d), and then the pigs were moved to a fattening house where they no longer received probiotics in the feed. At 115 ± 5 kg, the animals were transferred to the slaughterhouse. Analyses of carcass composition, muscle depth, protein content, total fat and ash, drip and cooking water loss, and fatty acids were performed. Pigs consuming the probiotic additives had improvements in some of the production parameters. According to our results, we could observe that some modifcations in meat composition after slaughter were observed in the group of pigs that consumed the probiotic additives during the postweaning, which could be considered as an improvement in meat quality. Pigs consuming probiotics had higher percentages of essential omega-3 fatty acids such as linolenic acid, and pigs consuming S. cerevisiae var. boulardii RC009 increased lean percentage and reduced the eicosanoic contents in meat. Also, an improvement in water retention capacity was observed in both groups treated with probiotics. While these results are promising, further studies are needed to evaluate the possible efect of these additives closer to slaughter, as well as their combined use.


Introduction
Pork is the most consumed meat worldwide, and consumers demand for safer pork is growing.Te purpose of the swine production chain is to provide food for people (fresh, cured, or processed) so that it necessarily involves aspects that infuence the pig's carcass quality.Te new trends in food production are based on the safety, innocuousness, and stability of animal origin products integrated in the concept "One World One Health" which maintains that human health and animal health are interdependent and linked to the health of the ecosystems in which they exist [1].Tus, the animal food is defned not only by its organoleptic attributes but also by the production process of these animals, involving health, welfare, and the environment.
Meat quality is a plural concept that difers depending on the segment of the meat chain.It can be related to its nutritional composition and organoleptic factors such as appearance, palatability, and safety.Consumers often agree that the most important quality criterion is the amount of muscle tissue (muscle) and the proportion of lean tissue [2].Since consumers demand lean meat, with fewer calories and cholesterol, the industrialist wants to pay for more muscle and less fat.Another very important parameter to analyse the quality of meat is the water retention capacity, intimately linked to the pH decrease after rigor mortis.It is also important to analyse the nutritional value given by the percentage of protein, the contribution of calories, and minerals [3].Although the benefcial and nutritional attributes of pigs are widely known, it should not be overlooked that the feed the animals receive can have an important infuence on meat quality, and although the interrelation with other aspects of the production process (genetics, management, and slaughter) should be considered, it is known that feed plays a fundamental role [4].Carcass composition traits have also been associated with the composition of the intestinal microbiota [5], which is closely related to the animal's diet and the way in which it can optimize the utilization of nutrients.
At the farm, the weaning is a very stressful moment because of many factors like the separation of their mothers, the change in the kind of feed and environment [6].Tese stress factors are also related to the occurrence of several intestinal and/or systemic pathologies that impact the farm performance indicators; their infuence on intestinal postweaning pigs gut health impacts on lifetime gut health and, consequently, on animal performance until slaughter [7].Tis encourages the use of antibiotics by producers to prevent the occurrence of diseases in the postweaning stage.It is known that the indiscriminate use of antibiotics generates resistance in pathogenic bacteria and represents a current threat to public health.An alternative developed in recent years to replace the nontherapeutic use of antibiotics and reduce the consequent impact on human health and the environment is the inclusion of probiotics as additives in the diet [8].
Méndez Palacios [9] concluded that there was an economic beneft when using both prebiotics and probiotics in the diet of pigs from weaning to fnishing.Tufarelli [10] found that feeding probiotic blend enhanced growth performance and meat quality in growing-fnishing pigs and also decreased faecal NH3-N and butyric acid levels, resulting in a viable approach to reduce animal excreta pollution.
Previous studies have demonstrated the probiotic properties of S. cerevisiae var.boulardii RC009 and P. pentosaceus RC007 isolated from the pig ecosystem.Tey were able to survive under gastrointestinal conditions and had benefcial properties such as aggregation/inhibition of pathogenic bacteria and the capacity to bind mycotoxins under gastrointestinal conditions [11,12].In addition, Poloni [13] showed that S. cerevisiae var.boulardii efectively counteracts the toxic efects of harmful afatoxin B 1 (AFB 1 ) in livers.However, they did not demonstrate the infuence of these strains on meat quality and other parameters related to lipid metabolism.Terefore, the study aimed to evaluate the inclusion of S. cerevisiae var.boulardii RC009 and P. pentosaceus RC007 as a probiotic additive in pigs' postweaning diets and its efect on meat composition, carcass characteristics, and fatty acids profle after slaughter.

Materials and Methods
Te working protocol and the used techniques comply with the regulations of the Subcommittee on Animal Bioethics under the Ethics Committee of Scientifc Research, as established in Resolution 376/22 of the Superior Council of the National University of Rio Cuarto.

Microorganisms.
Te probiotic yeast Saccharomyces cerevisiae var.boulardii RC009 was isolated from the pig intestine.Morphological, biochemical, and molecular characterization was conducted according to Armando [11].Species assignment was done using the Yeast Identifcation Database (https://www.yeast-id.com).Te sequence comparisons were performed using the basic local alignment search tool (BLAST) program within the NCBI database and submitted to GenBank (ID #KF447149.1).
Te probiotic lactic acid bacteria (LAB) Pediococcus pentosaceus RC007 was isolated from feedstuf and characterized based on morphological, physiological, and biochemical tests.DNA extraction, polymerase chain reaction, and 16S rDNA sequencing were performed using the method proposed by Martínez [12].DNA fragments were visualized after an electrophoretic run on 1.5% agarose gel stained with 0.5 μg ml −1 ethidium bromide, and gels were photographed using a MiniBIS Pro analyser (DNR Bio Imaging Systems, Jerusalem, Israel).Te fragment sizes were measured by comparison with DNA 100-bp ladder (Invitrogen by Life Technologies, Buenos Aires, Argentina).For DNA sequencing of both strands, template DNA was sent to Macrogen Inc. (Seoul, Korea).Sequences were compared using the local alignment search tool (BLAST) program with the NCBI database (GenBank) (http://www.ncbi.nlm.nih.gov/BLAST/) and submitted to GenBank (ID #1,980,444).Pediococcus pentosaceus RC007 culture conditions were 3 L of optimized culture medium developed with commercial refnery syrup, stirring 4 × g at 37 °C for 24 h, and 10% inoculum (v/v).Te concentration of dissolved oxygen at the beginning of the experiment was 0%.Foam production was controlled by the addition of antifoam 289 (Sigma-Aldrich, St. Louis, MO, USA).Te pH was maintained between 6.5 and 7 with the addition of 18 N H 2 SO 4 or Na 2 CO 3 20% w/v.

Probiotic Additives Formulation.
Te probiotic biomass obtained at the end of the fermentation of both microorganisms, LAB and yeast, was centrifuged at 1000 × g at 4 °C for 10 min.Te concentrated pellet was resuspended in the same volume of cryoprotectant (10% skim milk plus 5% yeast extract for the yeast and 10% skim milk only for LAB) and stored at −80 °C.Te viability of the lyophilized formula (1 g) was confrmed at the time of the trial.
2.4.Experimental Design.Tis experimental trial was carried out in the farm "Criadero de Cerdos de Aceitera General Deheza SA." Te studies were developed with male and female pigs of the commercial genetic line Agroceres PIC (Camborough × 337) in the breeding herd, which received the following vaccines: at 21 and 42 days of age, commercial PCVM vaccine (Circovirus + Mycoplasma) and at 50 days of age, A. pleuropneumoniae vaccine.Te animals were sexed and separated by weight similarity on the 1 st day of the test.For the experience, a total of 1650 animals were used (50% male and 50% female) which were housed in three diferent confned facilities.Te study was carried out with three consecutive weeks of weaning, and one treatment was applied to each of the weeks of weaning in weekly groups of 550 animals (full barn for each diet).From the frst day of the trial, pigs were fed ad libitum with diferent experimental diets.Treatments were applied after weaning (21 days of age) and for the total rearing period (until 70 days of age).In the rearing shed, foors were entirely plastic and the environment was fully controlled by tunnel ventilation with heating gas heaters.

Experimental Diets.
Commercial pig feed was the regular provided by the establishment where the trial was carried out.It covers the nutritional requirements of the rearing stage in all phases as recommended by the National Research Council [13].Te composition to each phase of diets is described in Table 1.Te phase 1 diet was administered from 21 days of age to 26 days of age, phase 2 diet from 26 to 34 days of age, phase 3 from 34 to 47 days, and phase 4 from 47 to 70 days of age.
In the treated diets, probiotic additives were incorporated in a concentration of 1 × 10 9 CFU/kg of feed, performing three treatments with 550 animals each; treatment 1: control diet (DC), treatment 2: DC with S. boulardii RC009, and treatment 3: DC with P. pentosaceus RC007.
After the essay, pigs were transferred to a fattening house where they consumed the regular fattening diet without the addition of probiotics.Once fnished the fattening stage (at 115 ± 5 kg and about 160 days hold), they were delivered to the slaughterhouse.

Carcass Composition and Muscle Tickness Determinations.
At the end of fattening, the pigs were sent to slaughter at approximately 160 days of age.In each treatment, 50 pigs (half male and half female) were randomly selected.Te transfer and slaughter protocol were the same for each treatment.Te carcass weight (kg) was determined at the time of slaughter, and after that, thickness of longissimus dorsi muscle measured between the 10th and 12th rib was recorded using a manual caliper.Also, a sample of 50 g of this region of the longissimus dorsi muscle in 50 pigs from each treatment was taken to make determinations in the laboratory.Two diferent formulas were applied to determine the percentage of lean: Fat o Meater (FOM): lean percent � 51.691 -0.214 × (A) -0.396 × (B) + 0.136 × (C); Hennesy: lean percent � 46.344 -0.580 × (B) + 0.232 × (C), where (A) is fat thickness in mm at 10 a rib, (B) is fat thickness in mm at last rib, and (C) is muscle thickness in mm.Veterinary Medicine International 3 2.7.Meat Quality Determination.Protein content, total fat, and total ash were determined by the AOAC [14] method.Drip loss analysis was performed according to the methodology described by Honikel [15].A meat fllet (20-50 g) was cut, weighed, hung on a hook, and placed in an infated plastic bag, taking care that the meat does not touch the bag.Te meat was conserved at 4 °C for 24 h, and the excess liquid was dried with flter paper and the fnal meat weight was taken.Te result was expressed as the percentage of moisture lost.Te assay was done in triplicate.
In addition, cooking loss was determined according to the methodology described by Rezar [16].Te same meat samples used for drip loss were used to determine water loss by cooking.Te samples were placed in plastic bags in a thermostatic bath at 75 °C.Te samples were cooked for 30 minutes and then removed from the bath and allowed to cool.Excess of moisture was removed with flter paper and the samples were weighed.Te moisture loss was expressed as a percentage.All samples were evaluated in triplicate.

Fatty Acids Analysis.
Te fatty acid profle of the meat samples from each treatment was determined.Fatty acid extraction was carried out following the methodology described by Ross [17] with some modifcations.Samples of approximately 3 g of meat were taken, lipid extraction was carried out using 30 mL of a 2 : 1 chloroform/methanol solution (v/v) for 30 min with refux, and saponifcation was carried out with 20 mL of 0.5 M NaOH (in methanol) and was refuxed at 100 °C for 20 min; 10 mL of hexane were added, and it moved to a decanter ampoule and the organic phase was taken.It was dried over anhydrous sodium sulphate and fltered with a 0.45 µm flter.Fatty acids were detected by gas chromatography using a gas chromatograph (GC) (Agilent 7890A, United States) equipped with a fame ionization detector (FID), an automatic injector (Agilent 7693A, United States), and a ZB-Wax column (60 m × 0.25 mm × 0.25 μm).Te chromatographic conditions were as follows: the injector and detector temperature were 250 °C, and helium was used as carrier gas with a linear velocity of 1 mL.min-1.Te gas chromatograph oven temperature was programmed to start at 180 °C (maintained for 1 min), followed by a 2 °C/min ramp to 240 °C.Te identifcation of fatty acid methyl esters (FAMEs) was achieved using a reference standard FAME mixture C14-C22 (Supelco Inc., Bellefonte, PA, USA).Fatty acids are expressed as a percentage of the sum of identifed fatty acids.All samples were evaluated in triplicate.

Carcass Weight and Muscle Tickness.
Table 2 shows the weight of the carcass in the treatments tested.Te pig carcass is the whole body of the slaughtered animal as it appears after bleeding and evisceration operations, split in half, without bristles, hooves, genitals, or diaphragm.Te results show that both carcass weight and muscle thickness are signifcantly improved in the probiotic treatments.

Meat Composition Determinations.
Te efects of different treatments on meat composition are reported in Table 3. Te total protein content in meat is an indicator of its nutritional value.Treatment with S. boulardii signifcantly increases the total protein content although this efect was not observed in the treatment with P. pentosaceus.Te mineral content (ash) of meat was not signifcantly diferent among treatment groups, and the meat intramuscular fat content decreased signifcantly in pigs consuming S. boulardii, while it increased in pigs consuming P. pentosaceus, with respect to the control group (P < 0.05) (Table 3).Te lean percentage in all treatments, both by the Fat O Meater method and by the Hennessy method, exceeded the 44% stipulated as the base value of lean tissue authorized for consumption according to the Secretariat of Agriculture, 4 Veterinary Medicine International Livestock, Fisheries, and Food (S.A.G.P. y A.). Te treatment with S. boulardii had the highest lean percentage of the study, being signifcantly higher (P < 0.001) than the control group (Table 4).

Water Retention Capacity Analysis.
Te loss of moisture by dripping and cooking was evaluated in samples from longissimus dorsi muscle from animals in the three treatments.Moisture loss by dripping was greater in the control group, with respect to the pigs that consumed the probiotic treatments that had higher water-holding capacity (WRC).Moisture loss by cooking was also signifcantly diferent between control samples and probiotic treatments, with diferences of about 30% less loss in the treated groups compared to the control (Table 5).

Fatty Acids Analysis.
Fatty acid profles and nutritional indices of the meat are presented in Table 6.Te SFA percentage was signifcantly diferent (P < 0.05) among the three groups, except for the C18 : 00 (stearic acid) content, which did not difer among the three groups.Te P. pentosaceus treatment resulted in signifcantly lower (P < 0.05) C14 : 00 percentage and no signifcant diferences compared to the control for C16 : 00 (palmitic) C22 : 0 (behenic).In contrast, S. boulardii treatment showed signifcantly lower (P < 0.05) C20 : 0 (eicosanoic) content.Te total MUFA content was signifcantly lower (P < 0.05) in the P. pentosaceus treatment, showing no signifcant differences (P < 0.05) from the control for the S. boulardii treatment.C18 : 1 (n-9) trans (elaidic) was the most abundant MUFA in all samples, with levels between 18.04 and 30.9%.Te total PUFA content was higher in the P. pentosaceus treatment.In all samples, C18 : 3 (n-3) cis (linolenic) was the major PUFA, with levels between 2.7 and 6.32%, whereas samples from pigs consuming P. pentosaceus had the highest content.Te P. pentosaceus treatment had a signifcantly lower (P < 0.05) total trans and cis fatty acid percentage.Te P. pentosaceus treatment resulted in a signifcantly lower (P < 0.05) content of trans and cis total fatty acids.For the control and P. pentosaceus treatments, no signifcant diferences were found between trans and cis total fatty acid percentage, whereas for the S. boulardii treatment, the proportion of cis total fatty acid was signifcantly higher (P < 0.05) than the trans total fatty acid.In addition, the S. boulardii treatment resulted in a signifcantly lower PUFA/SFA ratio (P < 0.05), and no diferences were found between the control and P. pentosaceus treatments.All treatments showed n-6 PUFA/n-3 PUFA ratios lower than the recommended value of 4, with P. pentosaceus treatment showing the lowest ratio.Of the meat health-promoting indices evaluated, only TI showed signifcant diferences between the probiotic treatments, being signifcantly lower for P. pentosaceus, although without diferences from the control.IA and NVI did not difer signifcantly among the three treatments.

Discussion
Pork quality is a broad concept that includes, among others, factors related to the nutritional and organoleptic characteristics of the product and its food safety.However, from an industrial point of view, several variables determine the degree of suitability of the product for industrial processing.In this study, pork composition, carcass characteristics, and fatty acid profle in slaughter pigs were evaluated after the inclusion of diferent probiotics in the postweaning phase.Te postweaning is recognized as one of the most challenging stages of pig growth that can afect their lifetime productivity [7,22].It was observed that the use of probiotic additives in the early growth stage had a positive infuence on production parameters such as carcass weight and muscle depth after slaughter.Tese improvements in growth performance with the addition of probiotics in feed have been previously reported [10,23,24] and were associated with improved intestinal health and utilization of nutrients released by the probiotic.Pork quality is afected by multiple factors throughout the meat chain.It is currently recognized that one of the most important quality criteria for the industry is the muscle percentage or lean production.In the present study, a signifcant increase in the proportion of lean meat was observed in pigs consuming S. boulardii, which is a desirable attribute for producers, consumers, and industrialists.Tese results are in agreement with Dávila-Ramirez [25], who observed an increase in lean percentage in pigs, under stress conditions, supplemented with S. boulardii.However, beyond the interest in obtaining lean meats, some authors indicated that genetic improvements to increase lean production in pigs lead to an unfavorable pattern of fatty acids in fat depots [26].Tis pattern is characterized by increased C18 : 2 n-6 trans ratios [27] as shown by our results in animals treated with S. boulardii.
Te World Health Organization recommends reducing the intake of saturated fats, which are associated with the production of cholesterol and the development of coronary heart disease.Instead, it recommends replacing them with the consumption of certain PUFAs that are considered essential, as they are not synthesized by the human body.In the present study, P. pentosaceus supplementation increased the levels of C18 : 3 (n-3) (linolenic) and consequently the total PUFA percentage.Tese results agree with those observed by Chang [28] and Grela [29] for the probiotic supplement in pig diets with Lactobacillus plantarum and a mixture of microorganisms (Lactococcus lactis IBB500, Carnobacterium divergens S1, Lactobacillus casei ŁOCK 0915, Lactobacillus plantarum ŁOCK 0862, and S. cerevisiae ŁOCK 0141, respectively).Te values obtained for the n-6 PUFA/n-3 PUFA ratio for the three treatments are low and are within the limits established by the World Health Organization to promote health and minimize cardiovascular disease risks.Zhimei [30] also obtained low values of the n-6/n-3 ratio for pigs treated with a control diet and supplemented with the probiotic L. reuteri 1 in pigs.On the contrary, Grela [27] and Chang [26] obtained very high values of this relationship for both, the control and supplemented with probiotics diet.Means in the same row with a common letter are not signifcantly diferent according to Fisher's protected LSD test (P > 0.05).Values are expressed as the means ± SD (n � 3).C14 : 0, miristic acid; C16 : 0, palmitic acid; C18 : 0, stearic acid; C20 : 0, arachidic acid; C22 : 0, behenic acid; C18 : 1 (n -9) trans, elaidic acid; C18 : 1 (n -9) cis, oleic acid; C18 : 2 (n -6) trans, linolelaidic acid; C18 : 2 (n -6) cis, linoleic acid; C18 : 3 (n -3) cis, linolenic acid.SFA, saturated fatty acid; UFA, unsaturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid.TI: thrombogenic index.AI: atherogenic index.NVI: nutritive value.

Veterinary Medicine International
Only for the S. boulardii treatment, signifcantly higher total trans fatty acid percentages than cis fatty acids were observed.Tis could be due to changes in the gut microbiota induced by the yeast modifying the biohydrogenation of PUFA or resulting in the inhibition of isomerases.
Te high SFA values, for C20 : 0 (arachidic acid), but particularly for the surprisingly high values of C22 : 0 (behenic acid), found in the study for the treatments have previously been linked to diet, genetic breed, and the weight and age of the animals.A diet rich in cereals and other carbohydrate-rich feeds may increase the levels of saturated fatty acids in meat [31].Pigs-fed diets high in corn or other grains tended to have more saturated fats.Some pig breeds tend to produce meat with higher saturated fat levels [32].Genetic makeup infuences fat deposition and metabolism in animals.In addition, heavier and older pigs tend to have a higher saturated fat percentage because they have more time to deposit fat in the body [33].However, since many of the variables were the same for all the groups under study, this diference in the formation and deposition of fatty acids in the muscle could be associated with modifcations in the intestinal microbiota as has been previously proposed [34].
Te nutritional value of pork meat can be improved by decreasing the SFA level because SFA are related to the incidence of cardiovascular heart diseases [35].In our study, we demonstrated that dietary supplementation with S. boulardii reduced the C20 : 0 (eicosanoic) amount in meat.Similar results were obtained by Zhu [36], who evaluated the efects of the addition of probiotics (a mixture of L. plantarum and S. cerevisiae) to the diet of Bama Mini-pigs on carcass traits and meat quality.In addition, the values obtained for the healthpromoting indices of meat were those expected for pigs TI � 1.12 and AI � 0.47 [37,38] and were similar to those obtained by Grela [29] for pigs fed with a control diet and supplemented with a combination of probiotics.On the other hand, there was a slight increase in the levels of trans fatty acids found in the pigs consuming the probiotics; it can be considered a negative efect from a health point of view.Further studies are needed to evaluate the signifcance of this efect.
In the present study, the ash content was not modifed in pigs receiving probiotics, in contrast to that reported by Chang [28] who found that dietary supplementation with probiotics signifcantly reduced the ash content although they also found an increase in the protein level.
For both the industry and the consumer of fresh pork, the water-holding capacity of muscle tissue has a central infuence.For the industry, loss of WRC reduces the sausage yield, while abundant loss of liquid during cooking generates consumer rejection.Tis loss of liquid retention capacity is associated with the rate of postmortem pH decrease.In our results, we observed that the water retention capacity was better in pigs that consumed the probiotic additive in the early stages of growth.Many causes associated with the management or slaughter can generate these pH changes.Although in the present study, the animals were slaughtered at the same age under the same protocol, and the possible efect of slaughtering with a period of 7 days between groups should be considered in the interpretation.Some modifcations in meat composition after slaughter were observed in the group of pigs that consumed the probiotic additives during the postweaning, which could be considered as an improvement in meat quality.While these results are promising, further studies are needed to evaluate the possible efect of these additives closer to slaughter, as well as their combined use.

Conclusion
Te group of pigs who were given probiotic supplements during their postweaning period showed some changes in their meat composition after slaughter, which may be interpreted as an improvement in the quality of pork.Pigs fed with probiotics showed higher percentages of essential omega-3 fatty acids, such as linolenic acid, and pigs fed with S. cerevisiae var.boulardii RC009 showed a decrease in eicosanoic contents in meat and an increase in lean percentage but it also increased the levels of trans fatty acids.In addition, both probiotic-treated groups showed an improvement in the capacity to retain water [39].

Table 1 :
Centesimal composition and calculated values of diets provided to the animals in the experimental period.

Table 2 :
Carcass weight and longissimus dorsi muscle depth measured between the 10th and 12th rib in pigs of the three performed treatments.

Table 3 :
Proteins content, intramuscular fat, and ash determinations of the three performed treatments.Values are expressed as a percentage of dry matter (DM).
aMeans in the same column with diferent superscripts (a and b) are signifcantly diferent (P < 0.001), according to Fisher's protected LSD test.Te standard deviation is enclosed in parenthesis.

Table 4 :
Lean percentage of the three performed treatments according to two diferent formulas: Fat o Meater (FOM) and Hennesy.Means in the same column with diferent superscripts (a and b) are signifcantly diferent (P < 0.001), according to Fisher's protected LSD test.Te standard deviation is enclosed in parenthesis.

Table 5 :
Drip loss and cooking loss analysis of the three performed treatments.

Table 6 :
Efects of probiotics addition in pigs' postweaning diets on fatty acid profle and contents in meat samples (relative % fatty acids).