Antioxidant Capacity of Lactic Acid Bacteria and Yeasts from Xinjiang Traditional Fermented Dairy Products

Abstract

(1) Background: The objective of this study was to screen strains with antioxidant potential from lactic acid bacteria (LAB) and yeasts isolated from traditional Xinjiang fermentation products. (2) Methods: Twenty-three strains of LAB and twelve strains of yeast isolated from traditional fermented dairy products from different regions of Xinjiang were selected, and the strains with antioxidant ability were initially screened by measuring the hydroxyl radical scavenging, superoxide anion scavenging, DPPH radical scavenging, ABTS⁺ radical scavenging, anti-lipid peroxidation, and ferrous ion chelating abilities of their bacterial bodies, cell-free extracts, and fermentation broth. They were further screened by measuring their superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities to identify strains with more powerful oxidative abilities. (3) Results: The results show that Lacticaseibacillus paracasei NM-12, Enterococcus faecium UM-12 and NM-11, and Pichia fermentans QY-4 had effective antioxidant enzyme activities. The SOD activity of these strains reached 4.846 ± 0.21 U/mL, 9.105 ± 0.428 U/mL, 8.724 ± 0.365 U/mL, and 6.518 ± 0.223 U/mL; the GPX activity reached 0.1396 ± 0.009 U/mL, 0.1123 ± 0.006 U/mL, 0.014 ± 0.007 U/m, and 0.0919 ± 0.006 U/mL; and the CAT activity reached 19.934 ± 3.072 U/mL, 3.749 ± 0.926 U/mL, 92.095 ± 1.017 U/mL, and 97.289 ± 0.535 U/mL, respectively (p < 0.05). (4) Conclusions: Lacticaseibacillus paracasei (NM-11), Enterococcus faecium (UM-12, NM-11), and Pichia fermentans (QY-4), isolated from traditional fermented dairy products, are probiotics with high antioxidant activity and potential applications in the food and fermentation industries.

1. Introduction

Probiotic foods are becoming increasingly popular in China and are widely claimed to be beneficial for health, thus driving their development. Probiotics are defined by the World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) as “Live microorganisms with probiotic benefits to the host when given in sufficient quantities” [1]. Increasing evidence, both nationally and internationally, supports the beneficial effects attributed to probiotics, including improved intestinal health, enhanced immune response, reduced serum cholesterol, and cancer prevention [2]. Probiotics have been found to exert antioxidant effects through their metal ion chelating ability, antioxidant systems, regulation of signaling pathways, and enzymes producing reactive oxygen species (ROS) to alleviate oxidative stress in the body [3,4].

Oxidative stress is an imbalance caused by an excess of ROS or oxidants that surpasses the body’s innate ability to mount an antioxidant response. Oxidative stress causes macromolecular damage and is associated with a variety of disease states, such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging [5,6]. Therefore, external antioxidants are needed to reduce oxidative stress. Currently, synthetic antioxidants have been extensively developed and added to foods, but these have been found to produce toxic side effects such as liver, spleen, and lung toxicity, and carcinogenesis [7,8]. Effective natural antioxidants seem to be healthier and safer than synthetic antioxidants [9]. Therefore, microorganisms have become one of the best choices for obtaining natural antioxidants due to their wide availability, low cost, and high safety.

El-Sayed et al. [10] studied the antioxidant capacity of several strains of Lactobacillus to enhance fermented camel milk and found that milk containing lactic acid bacteria had a higher antioxidant capacity during storage compared to milk containing commercial ferments. Kahar et al. [11] screened seven strains of yeasts with high antioxidant capacities and isolated Kluyveromyces marxianus and Saccharomyces cerevisiae from traditional fermented yogurt in southern Xinjiang. Abreu et al. [12] found that Saccharomyces boulardii improved the antioxidant defense of the kidney, restored serotonin and dopamine concentrations, and activated the renin angiotensin system (RAS) vasodilator and antifibrotic axis. Wang et al. [13], Brandão et al. [14], and Fang et al. [15] separately found that Limosilactobacillus reuteri, Lacticaseibacillus casei, and Lacticaseibacillus rhamnosus could lower cholesterol and reduce the atherosclerotic index while being safe and effective remedies for improving health, especially reducing the risk of cardiovascular disease. Therefore, screening bacteria from microorganisms that are more effective at relieving oxidative stress is of considerable research significance and presents substantial application prospects.

Xinjiang, located on the northwestern border of China, presents complex terrain, a diverse climate, many hours of sunshine, little rain, dryness, and strong ultraviolet radiation. Since ancient times, many ethnic groups have lived together in this area, and nomadic herding and agriculture have coexisted with long-established pastoral practices. The herders have accumulated rich experience in processing various dairy products, using traditional methods for their manufacture and achieving unique tastes and high nutritional value, thus conferring on traditional fermented dairy products unique local characteristics. We hypothesized that microorganisms in traditional fermented dairy products in Xinjiang would be able to tolerate oxidative stress due to their survival in this harsh environment under strong ultraviolet radiation. Therefore, we attempted to screen lactic acid bacteria (LAB) and yeasts with strong antioxidant capacities. LAB and yeasts isolated from traditional fermented dairy products of Xinjiang were screened to determine those with a strong antioxidant capacity based on their hydroxyl radical, superoxide anion, DPPH, ABTS⁺ radical scavenging, anti-lipid peroxidation, and ferrous ion chelating abilities and an antioxidant enzyme activity assay to provide a theoretical basis for the development and application of traditional fermented dairy products from Xinjiang.

2. Materials and Methods

2.1. Materials and Reagents

1,10-Phenanthroline hydrate, Nitroblue tetrazolium chloride (NBT), phenazine methosulfate (PMS), Iinoleic acid, NADH Na2, TBA (2-thiobarbituric acid), butylated hydroxytoluene (BHT), ascorbic acid (Vc), a Superoxide Dismutase (SOD) Activity Assay Kit, and a Micro Glutathione Peroxidase (GPX) Assay Kit were procured from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. 1,1–Diphenyl-2-picrylhydrazyl (DPPH) and ABTS diammonium salt were obtained from Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China. The Catalase (CAT) Activity Assay Kit was procured from Nanjing Jiancheng Bioengineering Institute. All chemicals and re-agents were of analytical grade (N99.7%).

2.2. Microorganisms

The strains selected for this study were lactic acid bacteria with a substantial probiotic effect, isolated and identified in our laboratory by Mairiyangu Yasheng and Yilimire Rexiati [16] from traditional fermented dairy products manufactured in different regions of Xinjiang, as well as yeasts isolated and identified from dairy products. All strains were stored at −80 °C in the Laboratory of Special Species Conservation and Regulatory Biology, College of Life Sciences, Xinjiang Normal University. Strain information is provided in Appendix A (

Table A1. Strain source information.

Number	Genus	Species	GenBank	Source
KM-12	Lactobacillus	Lactobacillus gallinarum	MW931798.1	Uruqmi yogurt pimple
KM-14	Lactobacillus	Lactobacillus gallinarum	MW931799.1	Uruqmi yogurt pimple
UM-2	Lactobacillus	Lactobacillus gallinarum	MW931811.1	Uruqmi cheese
TM-30	Lactobacillus	Lactobacillus gallinarum	MW931793.1	Tashkurgan County cheese
QM-27	Enterococcus	Enterococcus durans	MW931809.1	Tacheng cheese
NM-14	Enterococcus	Enterococcus durans	MW931841.1	Habahe County cheese
TM-1	Enterococcus	Enterococcus durans	MW931780.1	Tashkurgan County cheese
TM-7	Enterococcus	Enterococcus durans	MW931782.1	Tashkurgan County cheese
TM-25	Enterococcus	Enterococcus durans	MW931789.1	Tashkurgan County cheese
TM-28	Enterococcus	Enterococcus durans	MW931792.1	Tashkurgan County cheese
QM-5	Lacticaseibacillus	Lacticaseibacillus paracasei	MW931815.1	Tacheng cheese
KM-7	Lacticaseibacillus	Lacticaseibacillus paracasei	MW931821.1	Uruqmi yogurt pimple
NM-12	Lacticaseibacillus	Lacticaseibacillus paracasei	MW931840.1	Habahe County cheese
TM-26	Lacticaseibacillus	Lacticaseibacillus paracasei	MW931790.1	Tashkurgan County cheese
TM-27	Lacticaseibacillus	Lacticaseibacillus paracasei	MW931791.1	Tashkurgan County cheese
UM-12	Enterococcus	Enterococcus faecium	MW931812.1	Uruqmi cheese
UM-18	Enterococcus	Enterococcus faecium	MW931813.1	Uruqmi cheese
TM-3	Enterococcus	Enterococcus faecium	MW931781.1	Tashkurgan County cheese
TM-24	Enterococcus	Enterococcus faecium	MW931788.1	Tashkurgan County cheese
NM-11	Enterococcus	Enterococcus faecium	MW931839.1	Habahe County cheese
KM-6	Levilactobacillus	Levilactobacillus brevis	MW931796.1	Uruqmi yogurt pimple
AM-8	Lacticaseibacillus	Lacticaseibacillus rhamnosus	MW931827.1	Fuyun County cheese
AM-11	Lacticaseibacillus	Lacticaseibacillus rhamnosus	MW931830.1	Fuyun County cheese
QY-4	Pichia	Pichia fermentans	GU373759.1	Tacheng cheese
TY-20	Pichia	Pichia fermentans	MZ314865	Tashkurgan County cheese
QY-14	Geotrichum	Geotrichum candidum	MN736502.1	Tacheng cheese
NY-4	Yarrowia	Yarrowia lipolytica	KY110196.1	Habahe County cheese
TY-6	Yarrowia	Yarrowia lipolytica	MZ314862	Tashkurgan County cheese
TY-17	zeylanoides	Candida zeylanoides	MZ314864	Tashkurgan County cheese
2TY-1	Pichia	Pichia kudriavzevii	OM995975	Tashkurgan County cheese
TY-25	Guehomyces	Guehomyces pullulans	MZ314866	Tashkurgan County cheese
2TY-9	Wickerhamomyces	Wickerhamomyces anomalus	OM995983	Tashkurgan County cheese
4TY-6	Kluyveromyces	Kluyveromyces marxianus	OM995998	Tashkurgan County cheese
5TY-2	Issatchenkia	Issatchenkia orientalis	OM996002	Tashkurgan County cheese
6TY-2	Saccharomyces	Saccharomyces cerevisiae	OM996013	Tashkurgan County cheese

).

2.3. Instruments and Equipment

We used an Ultrasonic Homogenizer from Ningbo Scientz Biotechnology Co., Ningbo, China, LTD; a BIOTEK ELx808 Microplate Reader from the American Berten Instrument Co., Ltd., Ortenberg, Germany; a Hettich^® MIKRO 200/200R centrifuge from Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany; a vertical pressure steam sterilizer from Shanghai Shenan Medical Equipment Factory; and an electric constant- temperature water bath from Shanghai Yiheng Scientific Instruments Co., Shanghai, China.

2.4. Methods

2.4.1. Sample Preparation

The 23 strains of LAB were cultivated in MRS media at 37 °C for 24 h, while the 12 yeast samples were cultivated in YGC media at 28 °C for 24 h. After centrifugation at 4 °C and 10,000× g for 10 min, cells were collected. The supernatant was the fermentation broth needed for the experiment. The centrifuged bacteria were washed three times with phosphate-buffered saline (PBS) (pH 7.40) and then resuspended in PBS, and the bacteria count was adjusted to 10⁹ CFU/mL (OD600 nm ≈ 0.7) to obtain the bacteria for testing [17]. Then, bacteria with a cell density of 10⁹ CFU/mL were broken into cells using an ultrasonic ice bath (LAB: working time 6 s, interval time 9 s, 10 min, output power 200 W; yeast: working time 6 s, interval time 5 s, 10 min, output power 400 W) and centrifuged at 10,000 r/min, 4 °C for 10 min. The supernatant, a cell-free extract, was stored at −20 °C [18]. We used 2% L-Ascorbic acid/Vitamin C (Vc) as a positive control.

2.4.2. Hydroxyl Radical Scavenging Activity

The hydroxyl radical scavenging assay was carried out using the method described by Li et al. [19] and S. Shen et al. [20], with some modifications. We dissolved 5 mmol/L 1,10-phenanthroline (0.5 mL) and 7.5 mmol/L FeSO4 (0.25 mL) in pH 7.4 PBS (0.4 mL) and mixed thoroughly. Then, 0.01% H2O2 (0.25 mL) and sample fractions (0.5 mL) were added. The mixture was incubated at 37 °C for 1.5 h, and the absorbance was measured at 536 nm, with sterile water as a control. The results were determined using the following equation:

$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{y}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} (\%)=[1-\frac{A_2-A_1}{A_0}]\times 100$$

(1)

where A₁ is the absorbance of the sample, A₂ is the absorbance without H₂O₂, and A₀ is the absorbance of the control.

2.4.3. O²⁻ Radical Scavenging Activity

The O²⁻ radical scavenging activity was assessed using the non-enzymatic method of Goto et al. [21]. The sample solution (0.15 mL) was treated with 0.5 mL of 250 mmol/L PBS (pH 7.2), 2 mmol/L NADH (0.25 mL), and 0.5 mmol/L NBT (0.25 mL); after 5 min incubation at room temperature (approximately 22 °C) with 0.25 mL 0.03 mmol/L PMS, we measured the absorbance at 560 nm, with sterile water as a control. The O²⁻ radical scavenging activity was calculated using the following formula:

$${\mathrm{O}}^{2-} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y} (\%)=[1-\frac{A_1-A_2}{A_0}]\times 100$$

(2)

where A₁ is the absorbance of the sample, A₂ is the absorbance without PMS, and A₀ is the absorbance of the control.

2.4.4. DPPH Radical Scavenging Activity

The DPPH free-radical scavenging activity was determined using the protocol described by Goto [21] and Zhang et al. [22], with some modifications. DPPH was dissolved in ethanol absolute. Then, 0.2 mmol/L DPPH (0.5 mL) solution was mixed with 0.5 mL samples and incubated for 30 min at room temperature in darkness. The reaction tubes were centrifuged (4000 rpm, 10 min), and the absorbance was measured at 517 nm, with anhydrous ethanol as the control. The percentage inhibition of the DPPH free radicals was calculated using the following formula:

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} (\%)=[1-\frac{A_1-A_2}{A_0}]\times 100$$

(3)

where A₁ is the absorbance of the sample, A₂ is the absorbance without DPPH, and A₀ is the absorbance of the control.

2.4.5. ABTS⁺ Radical Scavenging Activity Assays

The ABTS⁺ radical scavenging activity assay followed the methods of Yang et al. [23] and Wang et al. [24], with modifications. We mixed 7 mmol/L ABTS⁺ and 2.45 mmol/L potassium persulfate in a 1:1 ratio and left the mixture to react in the dark for 12 h in order to generate ABTS⁺ radicals. Then, the mixture containing ABTS⁺ was diluted with PBS (pH 7.4) until reaching an absorbance level of 0.70 ± 0.02 at 734 nm. ABTS⁺ radical scavenging activity was evaluated by mixing 180 μL ABTS⁺ solution with 20 μL samples before incubation at 30 °C for 6 min. Then, the absorbance was recorded at 734 nm using a multifunctional microplate reader, with PBS as the control. The percentage inhibition of the ABTS⁺ radicals was calculated using the following formula:

$${\mathrm{A}\mathrm{B}\mathrm{T}\mathrm{S}}^{+} \mathrm{r}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} (\%)=[1-\frac{A_1-A_2}{A_0}]\times 100$$

(4)

where A₁ is the absorbance of the sample, A₂ is the absorbance without ABTS⁺, and A₀ is the absorbance of the control.

2.4.6. Lipid Peroxidation Inhibition Activity

The antioxidative activity was measured using the TBA method, based on monitoring the inhibition of linoleic acid peroxidation by the samples. The lipid peroxidation inhibition test followed the methods of Lin and Yen [25] and Chen et al. [26], with modifications. Twenty milliliters of linoleic acid emulsion was made with 1 mL of linoleic acid, 0.2 mL of Tween 20, and 19.7 mL of deionized water. PBS (pH 7.4) was mixed with 1 mL of linoleic acid emulsion, 0.2 mL of FeSO₄ (0.01%), 0.2 mL of ascorbate (0.01%), and 0.5 mL of the sample and then incubated at 37 °C. Deionized water was substituted for intracellular cell-free extract in the blank samples. After 12 h of incubation, 2 mL of the reaction solution was mixed with 0.2 mL TCA (4%), 2 mL TBA (0.8%), and 0.2 mL BHT (0.4%). This mixture was incubated at 100 °C for 30 min and allowed to cool. Two milliliters of chloroform was then added for extraction. The extract was obtained, and the absorbance was measured at 532 nm, with PBS as the control. The percentage of linoleic acid peroxidation inhibition was defined as follows:

$$\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}-\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d} \mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)=[1-\frac{A_1}{A_0}]\times 100$$

(5)

where A₁ is the absorbance of the sample and A₀ is the absorbance of the control.

2.4.7. Fe²⁺ Chelating Ability

The ferrous ion chelating ability test followed the methods of Amanatidou et al. [27] and Chen et al. [28], with modifications. We mixed 0.5 mL of the sample with 0.1 mL ascorbate (1% w/v), 0.1 mL FeSO4 (0.4% w/v), and 1 mL NaOH (0.2 M). Proteins in the cell-free extracts were precipitated by adding TCA (10% w/v), and the mixture was incubated in a water bath at 37 °C for 20 min. The supernatant was obtained via centrifugation at 3000× g for 10 min at 4 °C. Fe²⁺ chelating capacity in the de-proteinized supernatant was measured spectrophotometrically by determining the increase in absorption at 510 nm after a 10 min reaction with 0.5 mL 0.1% O-phenanthroline. Sterile water was used as a control.

$$\mathrm{F}\mathrm{e}\mathrm{r}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{s} \mathrm{i}\mathrm{o}\mathrm{n} \mathrm{c}\mathrm{h}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)=[1-\frac{A_1-A_2}{A_0}]\times 100$$

(6)

where A₁ is the absorbance of the sample, A₂ is the absorbance without FeSO₄, and A₀ is the absorbance of the control.

2.4.8. Antioxidant Enzyme Activity

The SOD, GSH-Px, and CAT in the intracellular cell-free extracts were evaluated spectrophotometrically using commercial assay kits following the manufacturers’ protocols. The enzyme activities are expressed as U/mL sample.

2.5. Statistical Analysis

All experiments were conducted in triplicate, and GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA) was used for data analysis. The results are expressed as the mean and standard error. The data were subjected to analysis of variance (ANOVA), and the differences between means were evaluated using Tukey’s test. Data were considered significantly different when the p-values were less than 0.05.

3. Results

3.1. Scavenging of Hydroxyl Radicals

Excessive hydroxyl radicals interfere with the normal metabolism of the body, causing oxidative damage to DNA, proteins, lipids, and other biomolecules, damaging normal cells and tissues, and accelerating the aging of the body, thus, inducing cardiovascular diseases, diabetes, tumors, and other diseases [29].

All the Lactobacillus fractions and yeast strains demonstrated hydroxyl radical scavenging activity, and most of the intact cell suspensions had a stronger hydroxyl radical scavenging ability than the cell-free extracts and fermentation broth groups. Furthermore, it has been observed that the antioxidant activity of these components may be influenced by the chemical composition present in MRS, YGC, and PBS, as evidenced by a comparison with the control group (Figure 1). The scavenging rates of hydroxyl radicals by the intact cell suspensions of each strain were 70%~99%, except for those of UM-2, QM-27, AM-11, TY-17, and 2TY-9. The clearance rates of other bacteria were higher than the control Vc (90.13%, p < 0.05). The clearance rates of 11 strains (KM-14, TM-1, TM-28, QM-5, TM-24, QY-4, QY-14, TY-6, 2TY-1, 4TY-6, and 6TY-2) were above 95% (p < 0.05), and the clearance rates of TM-1, TM-24, QY-4, and 2TY-1 were as high as 98.29%, 98.17%, 99.52%, and 99.08%, respectively.

The scavenging rate of hydroxyl radicals by the cell-free extracts of each strain ranged from 80% to 98%. The clearance rates of 13 LAB and 9 yeast strains were higher than the control Vc, with the hydroxyl radical scavenging rates of TM-1, TM-26, TM-27, and 2TY-9 being 96.28%, 97.33%, 97.81%, and 97.83%, respectively (p < 0.05).

The hydroxyl radical scavenging rate of the fermentation broth of each strain was 51%~99%. The clearance rates of eight LAB, namely UM-2, TM-1, TM-7, TM-25, TM-28, NM-12, TM-27, and AM-11, and all yeast strains were above 90.13%, with those of NM-12, QY-4, and 5TY-2 reaching 95.11%, 98.63%, and 99.03%, respectively (p < 0.05).

3.2. O²⁻ Radical Scavenging Activity

The dynamic balance of the redox system in living organisms can be disrupted by excessive superoxide anions (O²⁻), leading to oxidative stress and inducing a variety of diseases [28].

Both Lactobacillus and Saccharomyces fractions showed hydroxyl radical scavenging activity, but the intact cell suspension of Lactobacillus showed the highest superoxide anion scavenging rate, followed by the fermentation broth. The highest scavenging rate was observed for the cell-free extract of yeast, followed by the intact cell suspension (Figure 2). The superoxide anion scavenging rate of the intact cell suspensions of each strain was 23~37%. The clearance rates of 10 strains of LAB, including KM-12, KM-14, and QM-27, and 8 strains of yeast, including QY-4, TY-20, and QY-14, were higher than those of the control Vc (31.68%). The superoxide anion radical scavenging rate of TM-27, TM-3, AM-11, and TY-20 reached 34.66%, 34.12%, 36.76%, and 43.74%, respectively (p < 0.05).

The superoxide anion radical scavenging rate of the cell-free extracts of each strain ranged from 9% to 46%. The scavenging rate of the yeast cell-free extracts was significantly higher than that of the LAB (p < 0.05). For Lactobacillus, although the scavenging rates of all cell-free extracts were lower than the control, the scavenging rates of QM-27, NM-11, AM-8, and AM-11 reached over 20%. For yeast, except for 5TY-2 and 6TY-2, the cell-free extracts of all bacteria achieved a higher scavenging rate than the control, with the highest values (those of QY-4 and TY-20) reaching 45.35% and 45.19%, respectively (p < 0.05).

The superoxide anion scavenging rate of the fermentation broth of each strain ranged from 0% to 25%. The fermentation broth had a lower superoxide anion scavenging rate than the control Vc (31.68%). In contrast, the fermentation broths of Lactobacillus had higher scavenging rates compared to yeast, with 12 strains (KM-12, KM-14, UM-2, TM-30, QM-27, TM-1, TM-7, QM-5, TM-26, NM-11, 5TY-2, and 6TY-2) presenting scavenging rates of over 20% (p < 0.05).

3.3. DPPH Radical Scavenging Activity

DPPH, as a stable radical, is relatively simple to measure and easily reproducible. It contains a single electron, so it can accept one electron or hydrogen ion, and its maximum absorption is at a wavelength of 517 nm. In the presence of free-radical scavengers, the single electron of DPPH is trapped and causes its color to lighten and its absorbance value at the maximum light-absorption wavelength to decrease [30]. All fractions of LAB and yeast showed DPPH radical scavenging activity, as shown in Figure 3A. Both LAB and yeast fermentation broths showed a high DPPH radical scavenging rate; however, the MRS and YGC fermentation broth stocks also showed high scavenging rates, indicating that the chemical composition of the fermentation broth might have influenced its scavenging ability. In addition, the scavenging rates of both the intact cell suspensions and cell-free extracts were lower than that of the control Vc (91.16%) (p < 0.05).

The clearance rates of the intact cell suspensions of eight strains of LAB, including KM-12, KM-14, and UM-2, and nine strains of yeast, including QY-4, TY-20, and TY-6, were above 30%, with those of KM-14, TM-7, 2TY-1, and 6TY-2 reaching 33.03%, 33.16%, 46.82%, and 41.93%, respectively (p < 0.05).

Among the cell-free extracts, nine strains (KM-12, UM-18, TY-20, NY-4, TY-6, TY-17, 2TY-1, TY-25, 2TY-9, and 4TY-6) showed DPPH radical scavenging rates above 20%, with those of KM-12 and NY-4 reaching 29.94% and 26.39%, respectively (p < 0.05).

3.4. ABTS⁺ Radical Scavenging Activity

ABTS is widely used to determine the in vitro antioxidant capacity of a substance. ABTS reacts with K₂S₂O₈ to form a stable radical, ABTS⁺, which has a maximum absorption at 734 nm and appears blue-green in color. Thus, the antioxidant capacity can be judged [31].

Both LAB and yeast fractions showed ABTS⁺ radical scavenging activity (Figure 3B). As with the DPPH scavenging ability, the fermentation broths of Lactobacillus and the yeasts showed high scavenging rates, and the MRS and YGC stock solutions showed equally high scavenging rates. Thus, it could be assumed that the chemical composition of the cultures influenced the ABTS⁺ scavenging ability.

Regarding the intact cell suspensions, the clearance of the LAB UM-12, UM-18, and AM-11 was 29.53%, 27.08%, and 20.38%, respectively (p < 0.05). In contrast, the clearance of the yeasts was above 57%, with 6TY-2 reaching a maximum of 65.53% and QY-4, 2TY-9, and 4TY-6 reaching above 60% (p < 0.05).

Regarding the cell-free extracts, the clearance of Lactobacillus KM-14, NM-14, and TM-7 reached 42.71%, 40.27%, and 47.24%, respectively (p < 0.05). The clearance of all strains of yeast except TY-6 reached over 40%, with 5TY-2 and QY-14 showing the highest clearances of 55.53% and 53.08%, respectively (p < 0.05).

3.5. Lipid Peroxidation Inhibition Activity

Lipid oxidation often occurs during food processing and storage stages, which are associated with the formation of harmful compounds. During fat oxidation, fatty acids or fatty acyl side chains are attacked by chemical species and undergo a series of chain reactions with free radicals [32]. Therefore, the inhibition of lipid peroxidation can reduce economic losses and risks to human health in the food industry. All Lactobacillus and most yeast fractions showed resistance to lipid peroxidation. Among the Lactobacillus strains, most cell-free extracts demonstrated strong anti-lipid-peroxidation ability, as well as the intact cell suspensions and fermentation broths (Figure 4A, p < 0.05). Among the yeast strains, the fermentation broths demonstrated the strongest anti-lipid-peroxidation abilities, followed by the intact cell suspensions (Figure 4B, p < 0.05).

Regarding the intact cell suspensions, the anti-lipid-peroxidation rates of all seven strains, NM-12, TM-26, TM-27, UM-12, UM-18, KM-6, and AM-11, were higher than that of the control Vc (41.20%), with UM-12 reaching the highest rate of 50.60%. The anti-lipid-peroxidation rates of the yeast intact cell suspensions were all lower than that of the control Vc (41.20%), with TY-25 and 6TY-2 reaching 33.25% and 30.60%, respectively (p < 0.05). Regarding the cell-free extracts, 12 strains, including KM-12, KM-14, and QM-27, showed higher anti-lipid-peroxidation abilities than the control, with AM-11 presenting a rate of 57.10% (p < 0.05). In contrast, regarding the yeast cell-free extracts, only QY-4, TY-20, TY-6, and TY-17 demonstrated anti-lipid- peroxidation ability, with the rate of TY-6 reaching 27.47% (p < 0.05). Regarding the fermentation broths, the clearance rates of QM-5, AM-8, TY-20, QY-14, and 2TY-1 reached 42.62%, 41.45%, 54.46%, 53.49%, and 42.41%, respectively, which were all higher than that of the control Vc (41.20%) (p < 0.05).

3.6. Fe²⁺ Chelating Ability

Iron is the most abundant transition metal ion in the body, and under certain conditions it may catalyze the production of reactive oxygen species. The reduced form of iron enhances oxygen toxicity by converting less reactive hydrogen peroxide to reactive hydroxyl and ferric ions via the Fenton reaction. Therefore, excess iron is thought to contribute to cancer and cardiovascular disease [33]. Both LAB and yeast intact cell suspensions and cell-free extracts demonstrated ferrous ion chelating abilities, while the fermentation broths presented poorer chelating abilities than the negative control (original MRS and YGC culture broth) (Figure 5A,B). Regarding the intact cell suspensions, the chelation rates of KM-14, TM-30, TM-28, KM-7, TM-24, NM-11, and KM-6 were higher than those of the control Vc (74.80%), with KM-14 showing the highest chelation rate of 81.79% (p < 0.05). In contrast, the highest Fe²⁺ chelation rates of 52.38% and 54.53% were achieved by the intact yeast cell suspensions of TY-20 and TY-6 (p < 0.05). Among the cell-free extracts, UM-2 reached the highest chelation rate of 79.44%, surpassing that of the control Vc (p < 0.05). The chelation rates of NY-4 and TY-17 were the highest among the yeasts at 48.88% and 51.03%, respectively (p < 0.05).

3.7. Antioxidant Enzyme Activity

Cells possess a complex antioxidant system that regulates redox reactions through enzymatic and non-enzymatic processes to protect the organism from free radical damage. The main enzymatic systems are SOD, CAT, GPX, peroxide reductase, and glutathione sulfotransferase (GST). These enzymes are responsible for protecting cells from oxidative damage through effective interactions in the organism [34]. The results of the SOD activity measurements are shown in

Table 1. Determination of antioxidant enzyme activity.

Strain Number	SOD		GPX		CAT Vitality
	Inhibition Rate (%)	Vitality (U/mL)	Inhibition Rate (%)	Vitality (U/mL)	(U/mL)
KM-14	29.50 ± 0.90 cd	4.6545 ± 0.1995 cd	9.90 ± 0.99 efg	0.0340 ± 0.0034 efg	-
NM-14	39.55 ± 2.32 ab	7.3254 ± 07368 ab	-	-	19.7980 ±0.4009 d
TM-28	15.15 ± 0.60 e	1.9854 ± 0.0927 e	23.94 ± 2.82 de	0.0579 ± 0.0068 de	49.5629 ±2.5470 c
NM-12	30.35 ± 0.90 cd	4.8464 ± 0.2069 cd	34.45 ± 2.23 b	0.1396 ± 0.0090 b	19.9336 ± 3.0720 d
TM-27	40.88 ± 0.94 ab	7.6904 ± 0.2942 ab	-	-	16.3052 ± 1.8400 de
UM-12	44.99 ± 1.18 a	9.1054 ± 0.4284 a	31.73 ± 1.67 bc	0.1123 ± 0.0059 bc	3.7488 ± 0.9260 ef
UM-18	33.91 ± 0.86 bcd	5.7053 ± 0.2181 bcd	24.83 ± 1.34 bc	0.1260 ± 0.0068 bc	27.1753 ± 2.0760 d
TM-24	44.84 ± 1.34 a	9.0542 ± 0.4795 a	10.08 ± 1.46 ef	0.0409 ± 0.0059 ef	-
NM-11	43.95 ± 1.03 a	8.7241 ± 0.3646 a	4.08 ± 1.02 fg	0.0136 ± 0.0068 fg	92.0948 ± 1.0170 a
AM-11	28.21 ± 0.79 d	4.3687 ± 0.1717 d	58.82 ± 1.68 a	0.2383 ± 0.0068 a	-
QY-4	36.95 ± 0.79 bc	6.5178 ± 0.2234 bc	29.35 ± 1.88 cd	0.0919 ± 0.0059 cd	97.2890 ± 0.5346 a
TY-20	33.38 ± 3.84 bcd	5.6766 ± 0.9557 bcd	8.75 ± 2.50 fg	0.0238 ± 0.0068 fg	3.3724 ± 1.7850 ef
TY-6	35.10 ± 0.47 bcd	6.0116 ± 0.1247 bcd	22.73 ± 0.91 cd	0.0851 ± 0.0034 cd	-
TY-25	29.60 ± 1.44 cd	4.6842 ± 0.3294 cd	11.54 ± 1.67 ef	0.0409 ± 0.0059 ef	63.9560 ± 2.1760 b
6TY-2	27.67 ± 0.59 d	4.2520 ± 0.1239 d	27.27 ± 4.16 ef	0.1021 ± 0.0156 bc	14.8899 ± 2.1130 de
PBS	5.32 ± 0.32 e	0.6230 ± 0.0409 e	3.74 ± 0.94 fg	0.0255 ± 0.0034 fg	-

Different lowercase letters indicate significant differences between antioxidant enzymes of each strain (p < 0.05); the same letters indicate non-significant (p > 0.05).

. Strains NM-14 (39.55%), TM-27 (40.88%), UM-12 (44.99%), TM-24 (44.84%), NM-11 (43.95%), QY-4 (36.95%), and TY-5 (35.10%) demonstrated higher superoxide dismutase activity (p < 0.05). Regarding the results for GPX, strains NM-12 (34.45%), UM-12 (31.73%), AM-11 (58.82%), QY-4 (29.35%), and 6TY-2 (27.27%) showed higher glutathione peroxidase activity (p < 0.05). For CAT (Table 1), strains TM-28 (49.56 ± 2.54 U/mL), UM-18 (27.18 ± 2.08 U/mL), NM-11 (92.09 ± 1.02 U/mL), QY-4 (97.28 ± 0.53 U/mL), and TY-25 (63.96 ± 2.18 U/mL) showed higher peroxidase activity (p < 0.05).

4. Discussion

ROS in the body include superoxide anion radicals, hydroxyl radicals, and non-radical species of hydrogen peroxide, which not only cause oxidative damage in humans but also lead to food spoilage [35]. Therefore, the use of probiotics with antioxidant activity or products containing probiotics could effectively alleviate oxidative stress and some diseases in humans. In this experiment, all fractions of Lactobacillus and Saccharomyces exhibited favorable in vitro antioxidant effects. Most of the intact cell suspensions of Lactobacillus showed higher superoxide anion radical scavenging, DPPH radical scavenging, and ferrous ion chelation rates than the cell-free extracts and fermentation broths. The DPPH radical scavenging, ABTS⁺ radical scavenging, ferrous ion chelation, and anti-lipid-peroxidation rates were higher in the intact cell suspensions of yeast than in the cell-free extracts and fermentation broths, while the hydroxyl radical scavenging rates of most Lactobacillus and yeast fractions were very similar, indicating that most of the substances with antioxidant properties existed in the cells themselves, presenting antioxidant effects inside and outside the cells. Through the above six indexes, 10 strains of LAB and 5 strains of yeast with favorable in vitro antioxidant capacities were selected to determine their antioxidant enzyme activities, and most of the strains demonstrated high antioxidant enzyme activity.

Currently, natural antioxidants from dairy products are a hot research topic in China and abroad. Ren et al. [36] determined that the hydroxyl radical scavenging rates of the cell-free suspensions of nine Lactobacillus strains ranged from 10.37% to 94.26%, with the highest value of 94.26% achieved by strain CGMCC 1.557. Ding et al. [37] measured the antioxidant activity of LAB isolated from naturally fermented yak milk on the Tibetan plateau and found that Enterococcus faecalis achieved a hydroxyl radical scavenging rate of 86%, similar to that of most Enterococcus faecalis strains in this study (p < 0.05). Additionally, compared with the above studies, the LAB and yeast isolated from traditional fermented dairy products in Xinjiang demonstrated more substantial hydroxyl radical scavenging abilities. Chen et al. [28] measured the superoxide anion scavenging ability of intact cell suspensions of Pichia fermentans BY5 and Issatchenkia orientalis BY10 and found rates of 41.52% and 24.35%, respectively. The scavenging rates for Pichia fermentans (QY-4, TY-20) and Issatchenkia orientalis (5TY-2) determined in this study were not significantly different (p < 0.05).

Zhao et al. [38] and Huang et al. [39] determined the DPPH radical scavenging ability of LAB isolated from pickled vegetables and found that the intact cell suspensions of LAB had a stronger scavenging ability compared to cell-free extracts. These results are consistent with the results of the present study. Siesto et al. [40] measured the DPPH radical scavenging ability of intact cell suspensions of four Saccharomyces cerevisiae strains and showed that the scavenging rates ranged from 36% to 49%, demonstrating no significant difference compared to the rates obtained for 6TY-2 (Saccharomyces cerevisiae) and other yeasts in this study (p < 0.05). Zhang et al. [41] determined the ABTS⁺ free radical scavenging ability of 36 strains of LAB isolated from the intestines of hybrid groupers and found that the scavenging rate ranged from 6.53% to 28.98%. Pieniz et al. [42] examined the antioxidant activity of fermentation broths and cell-free extracts of Enterococcus species from meat and dairy products and found that the fermentation broth showed higher ABTS⁺ scavenging rates, which is consistent with the results of this study.

Ding et al. [43] and Han et al. [44] measured the anti-lipid-peroxidation capacity of several LAB species and found that intracellular cell-free extracts inhibited lipid peroxidation significantly more than intact cells (p < 0.05), which is consistent with our results for LAB. Jie et al. [45] determined the ferrous ion chelating ability of LAB isolated from traditional Sichuan kimchi and found that the chelation rate was higher in the intact cell and cell-free extract groups than in the fermentation supernatant group. Fakruddin et al. [46] determined the ferrous ion chelating ability of five strains of Saccharomyces cerevisiae isolated from fruits and found that their chelation rates ranged from 13.84% to 33.77%.

Wang and Li et al. [47] determined the SOD activity of LAB strains isolated from traditional Chinese dairy products, and the results showed that the inhibition rate ranged from 15.57% to 23.45%. Compared to the above-mentioned studies, most of the strains in the current study showed stronger SOD activity. To investigate the effects of Latilactobacillus curvatus (SR6) and Lacticaseibacillus paracasei (SR10-1) on sausage fermentation, Zhang et al. [23] determined their antioxidant activities and found that both strains had strong anti-DPPH radical scavenging, hydroxyl radical scavenging, and anti-lipid-peroxidation abilities, with CAT activities of 1.04 ± 0.87 U/mL and 1.17 ± 0.95 U/mL, respectively. However, these LAB did not exhibit T-SOD and GSH-Px activities, corroborating the Lactobacillus paracasei (TM-27) results obtained in the current study.

5. Conclusions

In this study, six in vitro antioxidant activity assays were used to determine the antioxidant activity of 23 strains of LAB and 13 strains of yeast isolated from traditional fermented dairy products manufactured in Xinjiang. Ten LAB and five yeast strains were initially selected based on their antioxidant activity. Subsequently, these strains were further screened through the determination of their antioxidant enzyme activity. The results indicated that NM-12 (Lacticaseibacillus paracasei), UM-12 (Enterococcus faecium), NM-11 (Enterococcus faecium), and QY-4 (Pichia fermentans) exhibited higher in vitro antioxidant and probiotic abilities. These strains provide a foundation for the subsequent development of functional antioxidant food ferments in the food fermentation industry, as well as the development of novel probiotic preparations.

The antioxidant activity in this study varied with the bacterial organism and the test method, so cell and animal tests should be conducted to verify the antioxidant properties of the strains. Furthermore, the LAB strains used in this study were identified as strains possessing favorable probiotic properties [16], while the probiotic properties of yeast strains have yet to be determined and require further investigation.