Re: 스피룰리나 발효 2025년

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Open AccessArticle

Optimized Spirulina Fermentation with Lacticaseibacillus rhamnosus: Bioactive Properties and Pilot-Scale Validation

by 

Akif Emre Kavak

 1,2,*

1

Department of Bioengineering, Faculty of Chemical and Metallurgical Engineering, Yıldız Technical University, Istanbul 34349, Turkey

2

Nuvita Biosearch R&D Center, Istanbul 34522, Turkey

3

Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Yıldız Technical University, Istanbul 34349, Turkey

4

Department of Industrial Engineering, Faculty of Engineering, Ondokuz Mayıs University, Samsun 55139, Turkey

5

Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering, Istanbul Technical University, Istanbul 34467, Turkey

*

Author to whom correspondence should be addressed.

Fermentation 2025, 11(5), 248; https://doi.org/10.3390/fermentation11050248

Submission received: 3 March 2025 / Revised: 17 April 2025 / Accepted: 25 April 2025 / Published: 1 May 2025

(This article belongs to the Special Issue 10th Anniversary of Fermentation: Feature Papers in Section "Industrial Fermentation")

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Abstract

Sustainable bio-based products derived from fermentation are gaining increasing interest. The present study was designed to determine the interaction of Lacticaseibacillus rhamnosus 23.2 bacteria with spirulina in a 3 L glass bioreactor and the effect of aeration and agitation speed on the final product biomass and antioxidant capacity. The fermentation medium contained only glucose, an inorganic salt mixture, and spirulina powder. The estimated biomass and antioxidant activity were found to be 3.74 g/L and 84.72%, respectively, from the results of the optimization model. Scale-up was performed with the obtained optimization data, and three pilot-scale fermentations were carried out in a 30 L stainless steel bioreactor. As a result of pilot production, the obtained bioactive products were freeze-dried, and their antibacterial, antioxidant, total phenolic properties, and cytotoxic activity were investigated. The pilot production results showed that the increase in bacterial cell number was around 3–4 log after 24 h of fermentation. An inhibitory effect against pathogenic bacteria was observed. A strong radical scavenging effect was found in antioxidant analyses. Total phenolic substance content was 26.5 mg gallic acid equivalent (GAE) g−1, which was the highest level in this study. Cytotoxic activity showed that bioactive products had a cytotoxic effect against Caco-2 adenocarcinoma cells. This study emphasizes the potential of Arthrospira platensis biomass as a substrate for the production of lactic acid bacteria (LAB)-based bioproducts. It is thought that the results obtained from this study may position potential innovative strategies in the food, pharmaceutical, agriculture, and cosmetic industries.

초록

발효를 통해 얻어진 지속 가능한 생물 기반 제품에 대한 관심이 

점점 증가하고 있습니다. 

본 연구는 3L 유리 바이오리액터에서 Lacticaseibacillus rhamnosus 23.2 균주와 

스피루리나의 상호작용을 조사하고, 

통기 및 교반 속도가 최종 제품의 생물량과 항산화 능력에 미치는 영향을 확인하기 위해 수행되었습니다. 

발효 배지에는 

글루코스, 무기염 혼합물, 스피루리나 분말만 포함되었습니다. 

최적화 모델의 결과에 따르면 추정된 생물량과 항산화 활성은 

각각 3.74 g/L와 84.72%로 나타났습니다. 

최적화 데이터를 기반으로 규모 확대를 수행했으며, 

30 L 스테인리스 스틸 생물반응기에서 3회의 파일럿 규모 발효를 실시했습니다. 

파일럿 생산 결과, 

얻어진 생물활성 제품은 동결건조되었으며, 

항균성, 항산화성, 총 페놀 성분, 세포독성 활성이 조사되었습니다. 

파일럿 생산 결과, 

발효 24시간 후 세균 세포 수가 약 3–4 log 증가했습니다. 

병원성 세균에 대한 억제 효과가 관찰되었습니다. 

항산화 분석에서 강한 자유 라디칼 소거 효과가 확인되었습니다. 

총 페놀 성분 함량은 

26.5 mg 갈산 등가량 (GAE) g−1로, 

본 연구에서 가장 높은 수준이었습니다. 

세포독성 활성 분석 결과, 

생물활성 제품은 Caco-2 선암 세포에 대해 세포독성 효과를 나타냈습니다. 

이 연구는 Arthrospira platensis 생물질을 

유산균(LAB) 기반 생물제품 생산의 원료로 활용할 수 있는 

잠재성을 강조합니다. 

본 연구 결과는 

식품, 제약, 농업, 화장품 산업에서 혁신적인 전략을 수립하는 데 

기여할 수 있을 것으로 기대됩니다.

Keywords: 

lactic acid bacteria; spirulina; bioreactor; bioprocess conditions; bioactive compounds

1. Introduction

The global food system places a heavy burden on the environment: greenhouse gas emissions, loss of biodiversity, destruction of terrestrial ecosystems, and is far from sustainable. Nowadays, it has been accepted that developing healthy, functional, and alternative bio-based products would be much better for a sustainable future, and fulfilling the goal is being carried out on bio-based products [1,2]. For that purpose, fermentation is one of the most extensively used technologies in the industry. Although fermentation dates back to ancient times, it is a technology that is still widely used in the present day. In fermentation, microorganisms assimilate the carbon and nitrogen sources and produce enzymes that catalyze the hydrolysis of sugars and proteins. These biochemical changes carried out by microorganisms during fermentation improve the functional value and nutritional properties of the products and directly contribute to the release of or change in bioactive compounds [3,4].

With approximately 260 species identified so far, lactic acid bacteria (LAB) are microorganisms widely distributed in milk, plants, meats, grains, and the gastrointestinal systems of vertebrates. LAB is one of the primary bacterial groups of industrial importance and is used in food production, health regulation, and the production of macromolecules, enzymes, and metabolites [5,6]. LAB species are cocci or rod-shaped, gram-positive, non-spore-forming, catalase-negative, non-cytochrome, non-aerobic but aerotolerant, acid-tolerant, and strongly fermentative bacteria that produce lactic acid as the primary end product during sugar fermentation. Examples of these bacteria include Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and Lactococcus species [7]. LAB species have the capacity to produce large quantities of bioactive compounds. Since their growth environments include food, dairy products, and plant-based foods, bioactive molecules such as bioactive peptides, polysaccharides, and bacteriocins are frequently found in fermented products [8,9,10].

Microalgae have become one of the most studied groups of organisms in recent years due to their rich content of proteins, fatty acids, vitamins, minerals, pigments, and many other valuable cellular metabolites [11]. Arthrospira platensis, a cyanobacterium (blue-green algae), is a phytoplanktonic organism suitable for intensive production. It is used as an alternative food source in human nutrition because it contains proteins, carotenoids, phycocyanin, chlorophyll pigments, vitamins, and oils. Spirulina contains high protein content, with 45% dry weight in samples and 62% in A. platensis cultured in the laboratory. More recent analyses have confirmed that the protein represents more than 60% and, in some cases, up to 70% of the dry weight. The protein content of spirulina is observed to be higher compared to other single-cell algae and cyanobacteria [12,13,14].

1. 소개

전 세계 식품 시스템은

환경에 심각한 부담을 주고 있습니다:

온실가스 배출, 생물 다양성 감소, 육상 생태계 파괴 등이며,

지속 가능성에서 멀어져 있습니다.

현재, 건강한, 기능적인, 대안적인 생물 기반 제품을 개발하는 것이

지속 가능한 미래를 위해 훨씬 더 나은 방법이라는 것이 인정되고 있으며,

이 목표를 달성하기 위해 생물 기반 제품 개발이 진행 중입니다 [1,2].

이를 위해 발효는

산업에서 가장 널리 사용되는 기술 중 하나입니다.

발효는

고대부터 사용되어 온 기술이지만,

현재까지도 널리 활용되고 있습니다.

발효 과정에서 미생물은

탄소와 질소 원료를 동화시켜

당과 단백질의 가수분해를 촉매하는 효소를 생산합니다.

미생물이 발효 과정에서 일으키는 이러한 생화학적 변화는

제품의 기능적 가치와 영양적 특성을 향상시키며,

생체활성 화합물의 방출이나

변화를 직접적으로 촉진합니다 [3,4].

현재까지 약 260종이 확인된 유산균(LAB)은

우유, 식물, 육류, 곡물, 척추동물의 소화계에 널리 분포하는

미생물입니다.

LAB는 산업적으로 중요한 주요 세균군 중 하나로

식품 생산, 건강 조절, 고분자, 효소, 대사산물 생산 등에 활용됩니다[5,6].

LAB 종은

구형 또는 막대형, 그람 양성, 포자 형성하지 않음, 카탈라아제 음성, 사이토크롬 음성, 혐기성이지만

혐기성 내성, 산성 내성, 강한 발효성을 가진 세균으로,

당 발효 시 젖산을 주요 최종 산물로 생성합니다.

이러한 세균의 예시로는

Lactobacillus,

Leuconostoc,

Pediococcus,

Streptococcus,

Lactococcus 종이 있습니다 [7].

LAB 종은

생물활성 화합물을 대량으로 생산할 수 있습니다.

그들의 성장 환경이 식

품, 유제품, 식물성 식품 등을 포함하기 때문에

발효 제품에서 생물활성 펩타이드, 다당류, 박테리오신과 같은

생물활성 분자가 자주 발견됩니다 [8,9,10].

미세조류는

단백질, 지방산, 비타민, 미네랄, 색소, 기타 다양한 가치 있는 세포 대사산물 함량이 풍부해

최근 몇 년간 가장 많이 연구되는 생물군 중 하나가 되었습니다 [11].

아르트로스피라 플라텐시스(Arthrospira platensis)는

청록색 조류(시아노박테리아)로,

집중 생산에 적합한 식물성 플랑크톤입니다.

인간 영양의 대체 식품원으로 사용되며,

단백질, 카로티노이드, 피코시아닌, 클로로필 색소, 비타민, 오일 등을 함유합니다.

스피루리나(Spirulina)는

건조 중량 기준으로 45%의 단백질 함량을 보이며,

실험실 배양된 A. platensis에서는 62%에 달합니다.

최근 분석 결과 단백질 함량이 60%를 초과하며,

일부 경우 최대 70%에 달하는 것으로 확인되었습니다.

스피루리나의 단백질 함량은 다

른 단세포 조류 및 청록색 조류에 비해 높은 것으로 관찰되었습니다 [12,13,14].

The beneficial effects of traditional fermented foods containing LAB on human health have been determined. Some of these benefits are related to protein-derived bioactive products. Protein-derived products produced by LAB include ribosomally produced and protein hydrolysate by-products used as natural preservatives and nutraceuticals. These protein-derived products with various application areas have attracted industrial attention [15,16]. LAB produces enzymes that catalyze the hydrolysis of proteins, thereby enabling the production of the amino acids they need. This ability they possess produces not only the free amino acids required by the bacteria but also a wide variety of peptides, some of which are equipped with biological activities. Each bacterial species has a different proteinase content that leads to a wide range of proteolytic activities, and the proteolytic activity occurs in a way that depends on the species and strain. Therefore, using lactic acid bacteria is an effective strategy for producing and eval‎uating bioactive peptides [17,18]. Obtaining valuable compounds as a result of the proteolytic activity of lactic acid bacteria is important for scientific studies to be carried out in this field. The LAB strains like Lactiplantibacillus plantarum, Levilactobacillus brevis, Lacticaseibacillus casei, Lactobacillus helveticus, Lacticaseibacillus rhamnosus, and Bacillus species have been the most commonly used starter cultures to obtain LAB fermented spirulina products with their probiotic properties and contributing to its chemical and functional properties [19,20,21]. Previous studies have shown that the products obtained as a result of the use of spirulina in LAB culture media have positive effects in terms of properties such as antioxidant, antimicrobial, taste, flavor, and number of viable cells [22,23,24]. In a study conducted by Niccolai et al. [25], Arthrospira platensis was fermented with Lactobacillus plantarum ATCC 8014. Not only the in vitro digestibility of fermented spirulina, but also the antioxidant and phenolic substance content were increased. As a result, it was reported that Arthrospira platensis biomass supports the growth and activity of probiotic bacteria during lactic acid fermentation and will be a potential substrate in the production of probiotic-based products. Similarly, Arthrospira platensis was fermented with Lactobacillus plantarum to improve its bioactive properties by De Marco Castro et al. [26]. Accordingly, at the end of the fermentation process, the total phenolic content was found to be 112%, and the DPPH radical scavenging capacity was found to be 60%. It was determined that the effects of fermented spirulina bioactive products increased compared to unfermented spirulina. In another study, Liu et al. [27] reported an increase in DPPH radical scavenging capacity after fermentation of A. platensis biomass in milk.

The present study aims to describe the addition of spirulina to the fermentation medium of L. rhamnosus, the optimization of bioprocess conditions, and to determine the effects of bioactive products. Optimization studies were carried out in a 3 L bioreactor with the help of a D-optimal experimental design. D-optimal design is generated by an iterative search algorithm and seeks to minimize the covariance of the parameter estimates for a specified model. In the fermentation optimization study, the effects on some operational parameters, including spirulina powder (g/L), aeration (vvm), and mixing speed (rpm), on the biomass and antioxidant capacity of the final product were examined. After finding the optimum medium composition, a scale-up was performed, and the study was carried out in a 30 L volume stainless steel bioreactor. Furthermore, as a consequence of pilot-scale studies of the bioactive product obtained, some properties were eval‎uated. This study is thought to be a guide for scale-up studies in new functional compounds development and fermentation.

LAB를 함유한 전통 발효 식품의 인간 건강에 대한

유익한 효과가 확인되었습니다.

이러한 효과 중 일부는

단백질 유래 생물활성 물질과 관련이 있습니다.

LAB에 의해 생성되는 단백질 유래 물질에는

리보솜 생성 단백질과 단백질 가수분해 부산물이 포함되며,

이는 천연 보존제 및 기능성 식품으로 사용됩니다.

다양한 응용 분야를 가진 이러한 단백질 유래 물질은

산업계의 관심을 끌고 있습니다 [15,16].

LAB는

단백질 가수분해를 촉매하는 효소를 생산하여

필요한 아미노산을 생성합니다.

이 능력은 박테리아가 필요로 하는

자유 아미노산뿐만 아니라 생물학적 활성을 갖춘 다양한 펩타이드를 생성합니다.

각 박테리아 종은

서로 다른 단백질 분해 효소 함량을 가지고 있어

다양한 단백질 분해 활성을 보이며,

이 활성은 종과 균주에 따라 달라집니다.

따라서

젖산균을 활용하는 것은

생물활성 펩타이드의 생산 및 평가를 위한 효과적인 전략입니다 [17,18].

젖산균의 단백질 분해 활성을 통해 얻어지는 가치 있는 화합물은

이 분야에서의 과학적 연구를 수행하는 데 중요합니다.

Lactiplantibacillus plantarum,

Levilactobacillus brevis,

Lacticaseibacillus casei,

Lactobacillus helveticus,

Lacticaseibacillus rhamnosus 및 Bacillus 속과 같은 LAB 균주는

프로바이오틱스 특성과 화학 및 기능적 특성에 기여하는

LAB 발효 스피루리나 제품을 생산하기 위해 가장 널리 사용되는 스타터 배양체입니다 [19,20,21].

이전 연구들은 스피루리나를 LAB 배지 매체에 사용해 얻은 제품이

항산화, 항균, 맛, 향, 생존 세포 수 등

다양한 측면에서 긍정적인 효과를 보였다고 보고했습니다[22,23,24].

Niccolai 등[25]의 연구에서

Arthrospira platensis는

Lactobacillus plantarum ATCC 8014와 발효되었습니다.

발효된 스피루리나의 체외 소화율뿐만 아니라

항산화 및 페놀성 물질 함량도 증가했습니다.

결과적으로 Arthrospira platensis 생물량은

젖산 발효 과정에서 프로바이오틱 박테리아의 성장과 활성을 지원하며,

프로바이오틱 기반 제품 생산의 잠재적 원료로 활용될 수 있다고 보고되었습니다.

De Marco Castro 등 [26]은

Arthrospira platensis의 생물학적 활성 특성을 개선하기 위해

Lactobacillus plantarum과 함께 발효시켰습니다.

이에 따라 발효 과정 종료 시 총 페놀 함량은 112%로,

DPPH 라디칼 소거 능력은 60%로 측정되었습니다.

발효된 스피루리나 생물활성 제품의 효과가

발효되지 않은 스피루리나에 비해 증가했다는 것이 확인되었습니다.

다른 연구에서 Liu 등 [27]은

우유에서 A. platensis 생물질을 발효한 후 DPPH 라디칼 소거 능력이 증가했다고 보고했습니다.

본 연구는

L. rhamnosus 발효 매체에 스피루리나를 추가하는 것,

생물공정 조건의 최적화,

생물활성 제품의 효과를 규명하는 것을 목적으로 합니다.

최적화 연구는

D-최적 실험 설계의 도움을 받아 3L 생물반응기에서 수행되었습니다.

D-최적 설계는 반복적 검색 알고리즘을 통해 생성되며,

지정된 모델의 매개변수 추정값의 공분산을 최소화하는 것을 목표로 합니다.

발효 최적화 연구에서는

스피루리나 분말 (g/L), 통기량 (vvm), 혼합 속도 (rpm) 등

일부 운영 매개변수가 최종 제품의 생물량 및 항산화 능력에 미치는 영향을 조사했습니다.

최적의 배지 조성을 확인한 후 규모 확대를 수행했으며,

연구는 30 L 용량의 스테인리스 스틸 바이오리액터에서 진행되었습니다.

또한, 얻어진 생체활성 제품의 파일럿 규모 연구 결과에 따라

일부 특성이 평가되었습니다.

이 연구는

새로운 기능성 화합물 개발 및 발효 분야의 규모 확대 연구에 대한

지침이 될 것으로 기대됩니다.

2. Materials and Methods

2.1. Isolation and Identification of Bacterial Strain

The homemade ripened cheese samples were collected from Elazığ Province, Turkey. Each sample was stored in a sterilized centrifuge tube at 4 °C and transported to the laboratory once collected. For the isolation and identification of lactic acid bacteria, 10 g of sample was taken from the isolation source, transferred to 90 mL of 0.9% NaCl solution, and homogenized for 2 min. After a series of dilutions with 0.85% (w/v) saline, these samples were spread on de Man, Rogosa, and Sharpe (MRS) agar [28] plates and incubated in an aerobic incubator (BD-S 56, Binder, Tuttlingen, Germany) at 37 °C for 48 h. Next, single colonies were picked from plates and cultivated in an MRS broth medium under 37 °C for overnight incubation. The morphology of strains was initially observed using a microscope to identify the strains that were obtained (CX23, Olympus, Tokyo, Japan). Details of the analyses performed for the identification of the strains are given in the Supplementary Materials. The strains were then further identified using 16S rRNA sequencing. Identified strains are listed in Supplementary Materials in Table S1. During the isolation LAB, 25 strains were identified as L. rhamnosus strains, and among them, L. rhamnosus 23.2 strain was selected to be used as a result of preliminary culture studies. The 16 S rRNA sequences of L. rhamnosus 23.2 strain identified in this study have been recorded in NCBI GenBank under the number PP843593.

2.2. Cultivation of Arthrospira platensis and Biochemical Composition

Arthrospira platensis (SP, 001) microorganism was obtained from the Nuvita Biosearch Center, Istanbul, Turkey. The components listed in the tables follow SAG Medium, which has been used for many years to grow spirulina. SAG Medium consists of Solution A, Solution B, P-IV Metal solution, and Chu micronutrient solution [29]. The contents of these components were given in Table 1, Table 2 and Table 3. After both Solution A and Solution B in Table 1 were sterilized in an autoclave (CL-40L, ALP, Tokyo, Japan), the contents of Solution B were transferred to the bottle containing Solution A and shaken until well mixed. The P-IV metal solution recipe was added to the nutrients in the listed order with constant stirring until approximately 950 mL of dH2O. The Na2EDTA was completely dissolved before adding the other components. Total volume was brought to 1 liter with dH2O. Chu micronutrient solution recipe, to approximately 900 mL of dH2O, was added each component in the order specified while stirring constantly. Total volume was brought to 1 liter with dH2O. [30]. All chemicals used in SAG Medium were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), and Isolab Chemicals (Istanbul, Turkey).

Table 1. SAG Medium Solution A and Solution B ingredients.

Table 2. P-IV Metal Solution Component.

Table 3. Chu Micronutrient Solution.

Spirulina was grown in 10 L glass bottles with 5% inoculum culture at 28 °C for 14 days. The strain culture was grown with continuous illumination at 200 µmol · m−2 · s−1 provided by warm white (3000 K) LED tube lamp (MAS LEDtube HF 600 mm HE 7W 840 T5, Philips) [31]. Concerning photoperiod, cycles of light:dark (h:h) (16:08) were followed. Culture, after reaching the 1 g/L dry weight, was harvested and dried in a freeze dryer to obtain powder form. Equation (5) was used to calculate the dry matter content.

Lyophilized A. platensis powder was analyzed for total carbohydrate, protein, lipid, moisture, and ash content. Total protein was analyzed following Lowry et al. [32]. Carbohydrates were quantified according to Dubois et al. [33], and lipids were determined according to Marsh and Weinstein [34]. Moisture and ash were determined by the method used by Inegbedion [35]. The biochemical composition of the biomass is given in Table 4. The plan of the study is shown in Figure 1.

스피루리나는 5% 접종 배양액을 사용해

10L 유리 병에서 28°C에서 14일 동안 배양되었습니다.

배양주는 따뜻한 백색(3000K) LED 튜브 램프(MAS LEDtube HF 600 mm HE 7W 840 T5, Philips)에서 200 µmol · m−2 · s−1의 연속 조명을 제공받아 배양되었습니다 [31].

광주기 조건은 빛:어둠 (h:h) (16:08) 사이클을 따랐습니다.

배양액은 건조 중량 1 g/L에 도달한 후 수확되어 동결 건조기에서 분말 형태로 건조되었습니다.

건조 물질 함량은 방정식 (5)를 사용하여 계산되었습니다.

동결 건조된 A. platensis 분말은 총 탄수화물, 단백질, 지질, 수분 및 회분 함량을 분석했습니다. 총 단백질은 Lowry 등 [32]의 방법을 따라 분석되었습니다. 탄수화물은 Dubois 등 [33]의 방법을 따라 정량화되었으며, 지방은 Marsh와 Weinstein [34]의 방법을 따라 측정되었습니다. 수분과 회분은 Inegbedion [35]의 방법을 사용하여 측정되었습니다. 생물량의 생화학적 구성은 표 4에 제시되어 있습니다. 연구 계획은 그림 1에 표시되어 있습니다.

Figure 1. Graphical plan of the study.

2.3. Optimization of Bioprocess Conditions 3 L Bioreactor

As a result of preliminary studies carried out on the Erlenmeyer scale, high-efficiency results in terms of outputs, such as the viable cell count and both wet and dry biomass in the fermentation process, were obtained in L. rhamnosus 23.2. Therefore, it was decided to use it in the current study. Effects of the parameters such as spirulina powder, aeration, and agitation were investigated on a 3 L bioreactor (Minifors 2, Infors HT, Bottmingen, Switzerland) with a working volume of 1.5 L at batch mode. For all the experiments, the pH was adjusted to 5.8, and the temperature was set to 37 °C. In all bioreactor experiments, 5 g/L of dextrose monohydrate and 2 g/L of inorganic salts were used in the culture medium as carbon source. Spirulina powder was pasteurized at 80 °C for 15 min and then added to the autoclaved bioreactor media. The composition of inorganic salt mix added to the fermentation medium at 2 g/L is as follows: CH3COONa 40%; K2HPO4 20%; C6H8O7·2NH3 15%; MgSO4·7H2O 15%; and MnSO4 10%. Oxygen tension was measured by determining the percentage of dissolved oxygen (DO%) relative to air saturation using an oxygen electrode (InPro 6830; Mettler Toledo, Switzerland). Fermentation time was set at 24 h and inoculation rate at 5%. Aeration rate (0–1 vvm), agitation speed (0–250 rpm), and spirulina powder (0–5 g/L) were used as optimization parameters. Agitation is crucial in overcoming mass transfer resistances in fermentation systems, and this is directly related to agitation speed. Aeration rate is an important process parameter for the growth of aerobic and anaerobic microorganisms [36,37]. For these reasons, they were selected to optimize fermentation in the study.

2.4. D-Optimal Experimental Design and the Optimization Phase

Design selection is a crucial concept in reaching the desired goals for an experimental study. A number of experimental designs are available in the current literature. This paper employs a D-optimal experimental design to construct a design matrix (DM) for the experimental study. The D-optimal design aims to maximize the determinant of the information matrix, and the exchange algorithm can be used to construct the DM [38].

We aim to optimize the lactic acid bacteria culture medium and find the highest antioxidant activity of the product to be obtained. Next, the design factors and collected experimental data are presented in Table 5. As shown in Table 5, the three design factors are specified as follows: A = spirulina powder (g/L), B = aeration (vvm), and C = agitation (rpm). Also, the two response variables are denoted as follows: y1 = biomass (g/L) and y2 = antioxidant activity (%). In Table 5, the DM of a D-optimal design consists of ten required design points, five lack-of-fit design points, and five replicate design points to carry out the experimental study for the responses, y1 and y2. The DM was constructed with the exchange procedure, and the computational time was 1266.57 s. Moreover, the experimental runs were randomly constructed without experimental bias.

The estimated biomass (g/L) response, 𝜇̂ 1(𝐱), is given as follows:

𝜇̂ 1(𝐱)=𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱 where 𝐱=⎡⎣⎢⎢𝐴𝐵𝐶⎤⎦⎥⎥, 𝐝=⎡⎣⎢⎢⎢⎢𝜙̂ 1𝜙̂ 2𝜙̂ 3⎤⎦⎥⎥⎥⎥, and 𝐃=⎛⎝⎜⎜⎜⎜⎜⎜⎜𝜙̂ 11𝜙̂ 21/2𝜙̂ 31/2𝜙̂ 12/2𝜙̂ 22𝜙̂ 32/2𝜙̂ 13/2𝜙̂ 23/2𝜙̂ 33⎞⎠⎟⎟⎟⎟⎟⎟⎟

(1)

where 𝜙̂ 𝑖 is the ith coefficient of the biomass regression function. Moreover, d and D represent the vector and matrix of the estimated regression coefficients for the biomass (g/L) response, respectively. Similarly, the estimated antioxidant activity (%) response, 𝜇̂ 2(𝐱), is acquired in the following way.

𝜇̂ 2(𝐱)=𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱 where 𝐱=⎡⎣⎢⎢𝐴𝐵𝐶⎤⎦⎥⎥, 𝐞=⎡⎣⎢⎢⎢𝜑̂ 1𝜑̂ 2𝜑̂ 3⎤⎦⎥⎥⎥, and 𝐄=⎛⎝⎜⎜⎜⎜⎜⎜𝜑̂ 11𝜑̂ 21/2𝜑̂ 31/2𝜑̂ 12/2𝜑̂ 22𝜑̂ 32/2𝜑̂ 13/2𝜑̂ 23/2𝜑̂ 33⎞⎠⎟⎟⎟⎟⎟⎟

(2)

where 𝜑̂ 𝑖 denotes the ith coefficient of the antioxidant activity regression function. Then, e and E are the vector and matrix of the estimated regression coefficients for the antioxidant activity (%) response, respectively.

A ratio of max to min greater than ten indicates a transformation. In Table 1, a ratio of max to min is 11.927 for the antioxidant activity (%) response, so a transformation is useful. The logit transformation technique was selected because the data were collected between 0% and 100% for the antioxidant activity (%) response. The transformation is obtained for the antioxidant activity (%) response as follows.

logit (𝑦2,𝑖)=ln(𝑦2,𝑖−𝑙𝑙𝑢𝑙−𝑦2,𝑖) and 𝑖=1, 2, …, 20

(3)

where lower limit (ll) and upper limit (ul) denote 0% and 100%, respectively.

The offered bi-objective optimization model aims to maximize the overall desirability function for the biomass (g/L) and the antioxidant activity (%) responses when dealing with boundary constraints. The overall desirability function is calculated using the geometric mean desirability functions of the biomass and the antioxidant activity. The bi-objective model is presented in the following way.

maximize (𝑑1(𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)∗𝑑2(Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)))1/2subject to −1≤𝐴,𝐵,𝐶≤+1where 𝑑1(𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)=⎧⎩⎨0, (𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)<𝐿1⎛⎝⎜⎜⎜(𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)−𝐿1𝑇1−𝐿1⎞⎠⎟⎟⎟, 𝐿1≤(𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)≤𝑇11, (𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱)>𝐿1,𝑑𝑐𝑟(Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱))=⎧⎩⎨0, Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)<𝐿2⎛⎝⎜⎜Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)−𝐿2𝑇2−𝐿2⎞⎠⎟⎟, 𝐿2≤Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)≤𝑇21, Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)>𝐿2,

(4)

In Equation (4), 𝑑1(𝜙̂ 0+𝐱′𝐝+𝐱′𝐃𝐱) and 𝑑2(Logit(𝜑̂ 0+𝐱′𝐞+𝐱′𝐄𝐱)) represent the desirability functions of the biomass and the antioxidant activity, respectively. Moreover, L1 and L2 are the lower values for the two responses. Further, T1 and T2 denote the specified target values for the two responses. In addition, the Design-Expert software (Ver. 12.0.3.0) was utilized for the experimental data analysis and plotting graphs. The MATLAB optimization toolbox (Ver. R2014a) was employed to acquire the optimum factor settings in (4).

2.5. Determination of Biomass

Dry cell weight is the weight of biomass calculated from the wet matter value in 1 g of sample in the moisture analyzer (Ohaus MB120, Parsippany, NJ, USA). The equation used is given below [39].

𝐷𝑟𝑦 𝑐𝑒𝑙𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (gL)=𝑊𝑒𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 (gL)·𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 (%)

(5)

2.6. Analyses of Antioxidant Activity

The DPPH (2,2-diphenyl-1-picrylhydrazyl) test is widely used to eval‎uate the capacity of antioxidants to neutralize free radicals. The basic principle of the test is that the purple color of DPPH turns yellow due to the antioxidant properties of the sample. In this test, 0.2 mM DPPH solution is mixed with the sample in a 9:1 ratio, and the analysis is performed. After incubation at dark for 30 min, absorbances were measured at 517 nm [40].

%𝐷𝑃𝑃𝐻=(𝐴𝑏𝑠𝐷𝑃𝑃𝐻−𝐴𝑏𝑠𝑆𝑎𝑚𝑝𝑙𝑒𝐴𝑏𝑠𝐷𝑃𝑃𝐻)×100

(6)

2.7. Pilot-Scale Production Experiments

After fermentation optimization, a scale-up study was carried out in a stainless steel stirred tank bioreactor with a working volume of 30 L (SKN-30, Kocaeli, Turkey). Since a scale-up factor of 1:10 was used, the study was switched from 3 L to 30 L. The bioreactor is made up of a glass vessel with four equally spaced vertical baffles and 12 cm diameter of stainless steel dual Rushon-style impellers that perform the agitation. The fermentation medium and the vessel were sterilized for 30 min at 121 °C, the pH of the medium was adjusted to 5.8, and the fermentation temperature was maintained at 37 °C. The working volume was determined to be 20 L, and pilot production runs were conducted three times for validation purposes. The culture media, 5 g/L dextrose monohydrate and 2 g/L inorganic salts, were used in pilot production. Additionally, optimum design factors were used with spirulina powder, and aeration and agitation were obtained as a result of the experiments carried out in a 3 L glass bioreactor. Additionally, a pilot production was conducted as the 4th study (without spirulina), where all conditions remained the same, and without spirulina powder in the medium. As a result of pilot production, the obtained products were lyophilized in a freeze dryer. Freeze-drying was performed for sublimation at −55 °C and a chamber pressure of 160 mTorr for 24 h and desorption for two h using the Epsilon 2–4 LSCplus freeze dryer (Martin Christ, Osterode, Germany). The freeze-dried bioactive product powders were packed using moisture barrier packages and stored at 4 °C until further analysis.

2.7.1. Monitoring Viable Cell Count

Fermentation was monitored by withdrawing samples from bioreactor productions at different time intervals, and the pour plate method [41] was used to enumerate viable cells. L. rhamnosus 23.2 colonies were counted in the MRS agar medium under aerobic conditions. Changes in the viable cell counts were determined as the log CFU/mL.

2.7.2. Antibacterial Activity

The antibacterial activity of the bioactive product was determined using the agar well diffusion method. Escherichia coli, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenese pathogenic bacteria were inoculated in Brain Heart Infusion broth (Neogen, Lansing, MI, USA). The density of lyophilized bioactive product and pathogenic bacteria was adjusted according to the McFarland (0.5%) standard. A 100 µL amount of each pathogen to be tested was spread on the BHI agar surface [42]. The wells (6 mm diameter) were prepared using a sterile well borer, and 100 μL of collected bioactive product was poured into the wells. The plates were then kept undisturbed for diffusion and incubated at 37 °C for 16–18 h. A clear zone of inhibition of 1mm or greater diameter (excluding 6 mm of well diameter) was considered positive inhibition, and actual values were given [43].

2.7.3. Antioxidant and Total Phenolic Content Determination

The total phenolic content assay was carried out according to Singleton et al. [44] using the Folin–Ciocalteu assay samples of 0.1 g of lyophilized bioactive product at 0 and 24 h dissolved in 10 mL of deionized water. To 100 μL aliquots of each sample, 2 mL of 2% sodium carbonate (Merck, Darmstadt, Germany) in water was added. After 2 min, 100 μL of 50% Folin–Ciocalteu reagent (Sigma-Aldrich, USA) was added. The reaction mixture was incubated in darkness at 25 °C for 30 min. The absorbance of each sample was measured at 760 nm using a UV–Vis spectrophotometric microplate reader (AMR 100, Allshengen, Hangzhou, China). Results were expressed in gallic acid equivalents (mg GAE g−1) through a calibration curve of gallic acid (0 to 500 μgmL−1) (Sigma-Aldrich, USA). Gallic acid calibration solutions of 0, 25, 50, 75, 100, 125, and 150 µg/mL concentrations were prepared in duplicates. Antioxidant activity was followed as described in Section 2.6.

2.7.4. Cytotoxic Activity

Cytotoxic activity was determined for bioactive compounds in each sample, and the modified method described by Cakir-Koc et al. [45] was used. Cytotoxicity was determined using Caco-2 adenocarcinoma cells. The Caco-2 cell line was obtained through the American Type Culture Collection (ATCC). Caco-2 cells were fed with 10% (v/v) fetal bovine serum (FBS) (PAN-Biotech, Aidenbach, Germany), 1% (v/v) non-essential amino acids (PAN-Biotech, Germany), and 1% (v/v) penicillin–streptomycin (PAN-Biotech, Germany) inactivated for one h at 56 °C in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (PAN-Biotech, Germany) containing 4.5 g/L glucose. Experiments were performed at 37 °C and in a 5% CO2:95% air atmosphere. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (AFG Bioscience, Northbrook, IL, USA) solution was then added to the wells after the medium parts of the wells were removed. The microplates were incubated at 37 °C for 4 h. After the incubation period, the optical density (OD) at 450 nm was determined using a microplate reader (AMR100 Microplate Reader, Hangzhou, China). The following formula (Equation (7)) was used to determine the number of viable cells.

Cell viability(%)=(AsAc)×100

(7)

where As is the absorbance of the peptide solution and Ac is the absorbance of the control sample.

2.8. Statistical Analysis for Pilot Production

The data were analyzed statistically using the SPSS Statistics software package (version 29.0, IBM Corp., Armonk, NY, USA). The quantitative data were analyzed using one-way analysis of variance (ANOVA) with the Duncan multiple range tests (p < 0.05). The total bacterial cell counts (CFU/mL) were converted to a logarithmic value before statistical analysis.

3. Results and Discussion

The study was initiated by identifying the bacterial strain and cultivating A. platensis. Co-cultivation of LAB and spirulina and optimization studies were carried out in a 3 L glass bioreactor with 20 different experiments. In consideration of the obtained optimization results, three different productions were carried out under the same conditions in a stainless steel bioreactor with a total volume of 30 L. Some tests were conducted to determine the properties of the bioactive products obtained as a result of pilot production.

The biochemical composition of powder form spirulina was analyzed and shown in Table 4. The protein content of spirulina used in the study was found to be approximately 60%.

Table 4. Biochemical composition of A. platensis biomass used in the study.

3.1. Experimental and Optimization Results of Bioprocess Conditions 3 L Bioreactor

Experimental design factors and responses for cultivation of L. rhamnosus in the 3 L bioreactor are given in Table 5. Pilot-scale validation studies were carried out with the optimum parameters obtained at the 3 L bioreactor scale.

Table 5. The three design factors and collected data for the experiment.

Table 6 shows the summaries of fits and ANOVA results of biomass (y1) and antioxidant activity (y2) responses. Also, the model reductions improve the models of y1 and y2 responses; therefore, the reduced cubic models (RCMs) are used in Table 6. Insignificant model terms were omitted for y1 and y2 responses, excluding the necessary ones to provide a hierarchy.

Table 6. Summaries of fits and ANOVA results of biomass (y1) and antioxidant activity (y2) responses.

The values of the maximum coefficient of determination (R2), the adjusted R2(𝑅2𝑎𝑑𝑗), and the absolute predicted coefficient of determination (𝑅2𝑝𝑟𝑒) are denoted in Table 6 for y1 and y2 responses. These values are good enough for y1 and y2 responses. Also, the differences were found to be less than 0.2 between 𝑅2𝑎𝑑𝑗 and 𝑅2𝑝𝑟𝑒 for each response. So, 𝑅2𝑝𝑟𝑒 values are in agreement with 𝑅2𝑎𝑑𝑗 values in Table 6. Next, adequate precision values measure the signal-to-noise ratios, and ratios of y1 and y2 responses are desirable when ratios are greater than or equal to four. For both responses, adequate signals were obtained. Then, Table 6 shows the F-values and p-values. Based on these values, the models of y1 and y2 responses were found to be significant. Further, the significant model terms are presented in Table 6 for y1 and y2 responses when using the RCMs. Moreover, the lack of fits was found to be non-significant for both responses, and indeed, the non-significant lack of fits was desirable. High R2 and 𝑅2𝑎𝑑𝑗 values are an indication of a close interaction between the experimental results and the values obtained from the model [46]. In the present study, from the high R2 and 𝑅2𝑎𝑑𝑗 values obtained, it can be said that the experimental and predicted values are very close to each other, indicating the success of the established model.

Based on Figure 2, the points were relatively close to the origin and distributed roughly symmetrically about the origin. Also, no patterns were observed in Figure 2. Thus, the RCMs are good fits for the experimental data.

Figure 2. Residual plots for (a) the antioxidant response and (b) the antioxidant activity response.

The RCMs of biomass (y1) and antioxidant activity (y2) responses were acquired in terms of coded equations in the following way.

𝜇̂ 1(𝐱)=6.40+4.16𝐵+8.00𝐶−7.92𝐴𝐵−6.24𝐴𝐶+2.01𝐵𝐶−1.10𝐴2              −9.64𝐴2𝐵−15.22𝐴2𝐶−17.33𝐴𝐵2+21.83𝐴𝐶2

(8)

𝜇̂ 2(𝐿𝑜𝑔𝑖𝑡(𝐱))=0.26+1.28𝐴−1.88𝐶+2.56𝐴𝐵+1.49𝐴𝐶−1.23𝐵𝐶−0.66𝐴2                         −0.79𝐵2−0.68𝐶2+1.74𝐴2𝐵+4.94𝐴2𝐶+4.87𝐴𝐵2−7.22𝐴𝐶2

(9)

The introduced bi-objective optimization model in Equation (4) was modeled with Equations (8) and (9). The aim of the bi-objective optimization model was to obtain the highest desirability function while acquiring the highest biomass and antioxidant activity. The optimum design factors are found as follows: A = 3.18 g/L, B = 0.93 vvm, and C = 119.97 rpm. The estimated biomass (g/L) and antioxidant activity (%) were found to be 3.74 and 84.72, respectively, from the results of the bi-objective optimization model. Also, the overall desirability value was acquired to be one. This desirability value indicates that the responses are highly desirable. Further, the validation run was carried out to verify the optimization results. As shown in Table 7, the actual biomass (g/L) and antioxidant activity (%) were acquired to be 4.23 and 81.59, respectively, from the validation study. Hence, it is reported that the bi-objective optimization and validation experimental results are in agreement to achieve the highest biomass and antioxidant activity.

Table 7. Results of optimization and validation run.

Figure 3 illustrates how the desirability and the estimated responses’ values change for the design factors using contour plots. As illustrated in Figure 3, the highest desirability, which was 1.000, was achieved as A = 3.18 g/L, B = 0.93 vvm, and C = 119.97 rpm. Also, the estimated biomass (g/L) and antioxidant activity (%) values may be observed in Figure 3, and the highest conditions are acquired as 3.74 g/L and 84.72%, respectively.

Figure 3. Contour plots for (a) spirulina powder (g/L) and aeration (vvm) design factors, (b) spirulina powder (g/L) and agitation (rpm) design factors, and (c) aeration (vvm) and agitation (rpm) design factors.

3.2. Pilot-Scale Production

Bioactive product production was carried out in a 30 L stainless steel bioreactor under optimized fermentation conditions in a 3 L glass bioreactor. Moreover, pilot-scale production was carried out. This production was repeated 3 times to include validation. The reason for replicating the 30 L pilot production studies three times was to validate pilot-scale production and demonstrate its feasibility. This is important because a major problem of pilot production in scaling-up studies is reproducibility. The products of pilot production studies were in powder form, obtained with a lyophilizer, and various analyses were performed. In the fourth study in pilot production, all parameters were kept constant, and without spirulina powder was not added.

3.2.1. Monitoring Viable Cell Count L. rhamnosus 23.2

The growth of L. rhamnosus 23.2 was monitored by taking samples at certain time points belonging to different bioreactor conditions during 24 h of fermentation. Figure 4 shows the viable cell counts of L. rhamnosus for pilot production. The results indicate that the presence of spirulina and fermentation conditions affect the growth of L. rhamnosus. In all four studies, cell counts were measured at close intervals initially. At the end of 24 h, the cell count of the fourth study was lower than the other three studies.

Figure 4. Variation in viable cell count for L. rhamnosus in 24 h pilot-scale production. Bars indicate mean and standard deviation.

At the initial stage of the pilot productions, the number of viable L. rhamnosus cells started with 4.5–6.5 log CFU mL−1 and increased to over 12 log CFU mL−1 as a result of 24 h of fermentation for three pilot productions. The highest number of viable cells was reached in the third study and at 24 h. The results of the three studies were close to each other. On the other hand, the number of viable cells without spirulina was lower than in fermented spirulina samples. Also, initially, without spirulina, the number of viable L. rhamnosus cells started with 5 log CFU mL−1 and increased to over 8 log CFU mL−1 as a result of 24 h of fermentation. The fourth study, without spirulina, was the production with the lowest number of viable cells. Previous studies have shown that the presence of spirulina in the fermentation medium increases the viable cell count of LAB. It was determined that the increase in the number of viable cells was directly proportional to the increase in biomass [47,48,49]. Spirulina powder is also important in increasing biomass. Spirulina protein content has been found to be between 55% and 70% in different studies. In the current study, spirulina powder with approximately 60% protein content was used [50,51]. Based on previous studies, higher viable cell counts were achieved in pilot-scale production than in bottle-scale production. Therefore, implementing processes at the pilot bioreactor scale, where aeration and mechanical agitation took place, potentially enhances the growth rate of the L. rhamnosus when compared to their flask-scale production.

3.2.2. Antibacterial Activity

The ability to inhibit the growth of pathogenic microorganisms is one of the mechanisms LAB performs to protect the host. Lactic acid bacteria’s proteolytic activity gives rise to small peptide compounds displaying antibacterial activity. These compounds are appreciated in industry productions as natural preservatives counteracting undesired contamination [52,53]. The results of the analysis of the antibacterial effects of bioactive products against the pathogens E. coli, S. aureus, S. enterica, and L. monocytogenes are given in Table 8. The third study showed inhibitory effects on all four different pathogens studied. In general, bioactive products showed inhibition against the L. monocytogenes and S. aureus pathogens. Inhibition against E. coli was observed in three pilot productions, especially the third study, which showed high efficacy. Similar results were obtained for S. enterica in three studies. When the results of the fourth study with and without spirulina were examined, it was found that there was no significant difference between fermented spirulina. Similar results were obtained when without spirulina was compared to fermented spirulina in Gram-negative bacteria. Numerous studies have demonstrated the antimicrobial activity and efficacy of lactic acid bacteria against foodborne pathogens [54,55]. Yang et al. [56] stated that when investigating the bacteriostatic mechanisms of the L. plantarum strain, which has significant antibacterial activity against L. monocytogenes, they found the highest antibacterial effect against L. monocytogenes among 12 different pathogenic bacteria. Tolpeznikaite et al. [57] stated that the antibacterial effect of fermented spirulina (FS) products was much better expressed against Gram-positive bacteria than against Gram-negative bacteria. They also reported the strong antimicrobial activity of FS against S. aureus. Lactic acid bacteria can inhibit the growth of pathogens in various ways, increasing the permeability of the thin outer membrane, altering the intracellular osmotic pressure, and inhibiting the synthesis of macromolecules. We suppose that the bioactive product may inhibit the expression‎ of L. monocytogenes membrane transport-related genes by producing bacteriocin production mechanisms, hence disrupting the cell membrane structure and inhibiting metabolic viability, biofilm, and growth.

Table 8. Antibacterial effect of bioactive products in mm (1st, 2nd, 3rd, and 4th study).

3.2.3. Antioxidant and Total Phenolic Content

Arthrospira platensis has attracted considerable attention due to its importance in terms of food and antioxidant properties. Fermentation may lead to spirulina products with better functional properties [58,59]. In the present study, the DPPH radical scavenging capacity of bioactive products at different fermentation times and different pilot production is shown in Figure 5. Radical scavenging capacity increased from the start (0 h) to 6 h of fermentation. From 12 h to 18 h, a general increase in radical scavenging capacity in three bioactive products was observed. It is worth pointing out that after 24 h of fermentation, three bioactive products still contain high DPPH radical scavenging capacity (>70%). When Figure 5 is examined, it was determined that fermented spirulina studies showed higher antioxidant scavenging activity compared to without spirulina (fourth). It is thought that the secondary metabolites contained in spirulina have a positive effect on antioxidant activity during the fermentation process. The findings also support the results in the literature. At the end of fermentation, the highest antioxidant capacity was obtained in the third study. Even though the specific antioxidant mechanism of LAB remains unclear, we have found that LAB can produce antioxidant metabolites and scavenge reactive oxygen species (ROS) enzymes. LAB strains upregulate the activity of host antioxidant enzymes, downregulate the activity of enzymes related to ROS production, and regulate the antioxidant signaling pathway in hosts and intestinal flora. Fermentation with selected strains of different LAB strains was attempted by Yay et al. [24], Jamnik et al. [23], de Marco Castro et al. [26], Niccolai et al. [25], and Liu et al. [27] to increase their antioxidant properties and determine positive results. Different studies show that the presence of spirulina in an LAB medium contributes positively to its bioactive properties. Furthermore, the functionality of protein hydrolyzates may be diverse between strains due to the presence of different proteolytic systems in different microorganisms [60,61,62]. The strong antioxidant effect of lactic acid bacteria was proven by DPPH analysis in the study of Das et al. [63].

Figure 5. Antioxidant capacity expressed as mg of gallic acid equivalent (GAE) per g of bioactive products for three pilot productions. Bars indicate mean and standard deviation.

In the present study, DPPH radical scavenging capacity and total phenolic content results were determined similarly in pilot productions. The total phenolic content results are given in Figure 6. The total amount of phenolic compounds in all samples was expressed as mg/L−1 of gallic acid equivalent. The highest total phenolic content of 26.5 mg GAE g−1 was obtained in the third pilot production at 24 h. They were found, 24.5 and 25 mg GAE g−1, first and second studies, respectively. In pilot validation studies, total phenolic content was found to be higher at 18 and 24 h. The total phenolic content without spirulina was found to be lower than that of fermented spirulina studies. Phenolic compounds are considered major contributors to antioxidant capacity [64]. The total phenolic content of spirulina varies from 5 to about 50 mg GAE g−1, depending on strain, culture, and fermentation conditions [22,65,66]. According to Filannino et al. [67], the strain-specific metabolism of phenolic acid derivatives by lactic acid bacteria is strongly dependent on the intrinsic factors of the substrate. Phenolic compounds are one of the main factors responsible for the biological activity, with phycocyanin released from A. platensis biomass and compounds with antioxidant potential. In addition, the total phenolic compound of spirulina may be affected by cultivation conditions such as pH, light, temperature, and the downstream process. Moreover, the fermentation process could have released polyphenols such as gallic acid, converted phycocyanins to phycocyanobilin, or produced other metabolites, enhancing the antioxidant, and this shows the positive effect of using spirulina in an LAB culture medium [68,69]. Spirulina activates cellular antioxidant enzymes, prevents lipid peroxidation and DNA damage, scavenges free radicals, and increases catalase activity. Thus, it contributes positively to antioxidant and total phenolic substance content [70].

Figure 6. Total phenolic content expressed as mg of gallic acid equivalent (GAE) per g of bioactive products for three pilot productions. Bars indicate mean and standard deviation.

3.2.4. Cytotoxic Activity

Cytotoxic effects were detected in bioactive products for the analysis of bioactivity in pilot production. The results are given in Figure 7. No statistical difference was found between the concentration of the control sample and the lowest concentration of the first study (1.25 mg protein/mL) (p > 0.05). Cell viabilities ranged between 86.94 and 88.65%, 82.13 and 85.14%, and 76.11 and 78.46% for 1.25, 2.5, and 3.75 mg protein/mL, respectively. The highest inhibition was detected for all studies at the highest sample concentration (12.5 mg protein/mL). Cell viability was found to be 47.19%, 48.73%, and 49.16% at this concentration for the third, second, and first study, respectively. In the study without spirulina, the highest inhibition was detected at a concentration of 12.5 mg protein/mL. The cytotoxic effect without spirulina was found to be less than that of fermented spirulina. Ozturk et al. [71] showed that FS products of K. marxianus and L. helveticus and their unfermented spirulina counterparts did not reduce HUVEC and RAW264.7 cell viability below 60%, and the bioactive products could be considered biocompatible. Rosa et al. [72] determined that low concentrations of probiotic whey beverages had no effect on the PC-3 cell line, but had an antiproliferative effect at high concentrations. Yay et al. [24] reported that L.helveticus and K.marxianus spirulina fermentation resulted in a screening that revealed that “cascade” FS significantly decreased the viability of colon cancer cells (HT-29) in a dose-dependent manner, with up to a 72% reduction. Furthermore, doses ≤ 500 μg/mL−1 of “cascade” FS proved safe and effective in suppressing nitric oxide release without compromising cellular viability. Kayacan-Cakmakoglu et al. [73] studied the cytotoxic effects of bioactive peptides obtained by yogurt fermentation. They found that bioactive peptides had antiproliferative effects against Caco-2 cells. Grover et al. [74] reported that C-phycocyanin, a pigment obtained by the extraction of spirulina, had a strong immunomodulatory effect in a study conducted on an animal model and also did not have a cytotoxic effect. Current study findings emphasize the distinct and enhanced efficacy of pilot-scale production in exerting cytotoxic effects on cancer cells, context of colon cancer, presenting a potential avenue for further exploration of this fermented product in terms of its bioactive compounds. It is thought that phytopigments (carotenoids, chlorophyll, phycocyanin) and polysaccharides contained in spirulina contribute to the cytotoxic effect.

Figure 7. Cell viability (%) of Caco-2 cells treated with different concentrations of bioactive products. 0 represents the control cells, and other samples show the concentration of the bioactive product applied. A–E Different uppercase superscript letters in the same concentration indicate differences between samples. a–e Different lowercase superscript letters in the same sample indicate differences between samples. Bars indicate mean and standard deviation.

4. Conclusions

This study highlighted the importance of bioactive compound production by the addition of spirulina to the fermentation medium of L. rhamnosus, one of the LAB species, and the particular findings of the pilot-scale bioreactor study. Tests performed after pilot-scale production showed strong antioxidant and total phenolic content. Inhibition against pathogenic bacteria eval‎uations are an important indicator for the use of bioactive products in industry. Furthermore, in cytotoxic activity studies, bioactive products were found to have antiproliferative effects against Caco-2 cells. Pilot studies of fermented spirulina have determined that it exhibits higher bioactive properties compared to without spirulina. A lot of studies have already shown that the lactic acid fermentation of A. platensis improves its functional value. Hence, the aim of our study was to produce fermented A. platensis biomass on a pilot scale by scaling up and to further eval‎uate the potential of this bioactive product to be introduced to the commercial market. Considering the commercial importance of current LAB and microalgae products, there is a greater need to optimize microbial fermentation processes and pilot-scale production studies. To our knowledge, between LAB and spirulina, pilot-scale co-cultivation studies have never been conducted so far. In this respect, our study is original and provides significant gains to the literature.

4. 결론
이 연구는 LAB 종 중 하나인 L. rhamnosus의 발효 매체에 스피루리나를 추가함으로써 생물활성 화합물 생산의 중요성을 강조했으며, 특히 파일럿 규모 생물반응기 연구의 특이한 결과를 제시했습니다. 파일럿 규모 생산 후 수행된 시험 결과, 강력한 항산화 활성과 총 페놀 함량이 확인되었습니다. 병원성 세균에 대한 억제 효과 평가는 생체활성 제품의 산업적 활용을 위한 중요한 지표입니다. 또한 세포독성 활성 연구에서 생체활성 제품은 Caco-2 세포에 대한 증식 억제 효과를 나타냈습니다. 발효 스피루리나에 대한 파일럿 연구는 스피루리나를 첨가하지 않은 경우보다 더 높은 생체활성 특성을 갖는 것으로 확인되었습니다. 많은 연구에서 A. platensis의 젖산 발효가 그 기능적 가치를 향상시킨다는 것이 이미 입증되었습니다. 따라서 본 연구의 목적은 발효 A. platensis 생물질을 파일럿 규모로 확대 생산하고, 이 생물활성 제품의 상업적 시장 도입 가능성을 추가로 평가하는 것입니다. 현재 LAB 및 미세조류 제품의 상업적 중요성을 고려할 때, 미생물 발효 공정 최적화와 파일럿 규모 생산 연구의 필요성이 더욱 강조되고 있습니다. 우리 지식 범위 내에서 LAB과 스피루리나 간의 파일럿 규모 공배양 연구는 지금까지 수행된 적이 없습니다. 이 점에서 본 연구는 독창적이며 문헌에 중요한 기여를 제공합니다.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11050248/s1, Table S1: Identified LAB strains

Author Contributions

Study conception and design: A.E.K. and D.B.; data collection and performance of the experiments: A.E.K., A.Ö. and E.D.; analysis and interpretation of results: D.B. and O.S.; draft manuscript preparation: A.E.K., E.D., A.Ö., D.B. and O.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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