Re: 피스타치오 발효산물 - bioactive peptide...

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Bioactive peptides released by lactic acid bacteria fermented pistachio beverages

Author links open overlay panelSerena Marulo a, Salvatore De Caro a, Chiara Nitride b, Tiziana Di Renzo a, Luigia Di Stasio a, Pasquale Ferranti a b, Anna Reale a, Gianfranco Mamone a

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https://doi.org/10.1016/j.fbio.2024.103988Get rights and content

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Highlights

• •
• •
• •
• •
• •

• 발효된 피스타치오 음료에서 단백질 분해가 일어났습니다.
• •방출된 펩티드의 대부분은 2S 알부민, 7S 및 11S 글로불린에 속합니다.
• •내인성 프로테아제 외에 랩 프로테아제가 펩티드 분해에 관여합니다.
• •발효 과정에서 잠재적인 생체 활성 펩티드가 방출됩니다.
• •음료의 펩티드 프로필은 발효하는 랩에 따라 다릅니다.

Abstract

Fruit and vegetable are the raw materials for many popular fermented beverages with potential health benefits. In the present study, a novel pistachio beverage fermented with selected strains of lactic acid bacteria belonging to the species Leuconostoc pseudomesenteroides and Companilactobacillus paralimentarius was produced, and the extent of proteolysis was eval‎uated. Physico-chemical and microbiological analyses, together with peptidomics and proteomics, were carried out on the beverage after 24 h of fermentation, using both the non-inoculated and chemically acidified beverage as controls. Amino acid sequence of hundreds of peptides mainly released from 2S albumin, 11S and 7S globulin were characterized. The number and frequency of identified peptides were higher in beverage started with Leuconostoc pseudomesenteroides, followed by Companilactobacillus paralimentarius and acidic beverage. These results suggest that, in addition to endogenous proteases active at acidic pH, the proteolytic system of LAB directly participated to peptide degradation to some extent. According to the BIOPEP database, a group of 31 peptides were potentially bioactive, and primarily associated with antioxidative properties, ACE and DPP-IV inhibition. The beverage started with Leuconostoc pseudomesenteroides showed the highest amount and number of bioactive peptides. This study lays the foundations for the design of novel pistachio-fermented beverage with potential health properties.

초록

과일과 채소는

건강에 유익한 것으로 알려진 많은 인기 있는 발효 음료의 원료입니다.

본 연구에서는

Leuconostoc pseudomesenteroides와 Companilactobacillus paralimentarius 종에 속하는

유산균의 선택된 균주로 발효시킨 새로운 피스타치오 음료를 생산하고

단백질 분해 정도를 평가했습니다.

발효 24시간 후

음료에 대해 물리화학적 및 미생물학적 분석과

펩티도믹스 및 프로테오믹스 분석을 실시했습니다.

대조군으로 무접종 음료와 화학적으로 산성화된 음료를 사용했습니다.

2S 알부민, 11S 및 7S 글로불린에서 주로 방출되는

수백 개의 펩티드의 아미노산 서열이 특성화되었습니다.

확인된 펩타이드의 수와 빈도는

Leuconostoc pseudomesenteroides로 시작하는 음료에서 더 높았고,

그 다음으로 Companilactobacillus paralimentarius와 산성 음료 순이었습니다.

이 결과는

산성 pH에서 활성화되는 내인성 프로테아제 외에도,

LAB(lactic acid bacteria)의 단백질 분해 시스템이

어느 정도 펩타이드 분해에 직접 관여했음을 시사합니다.

BIOPEP 데이터베이스에 따르면,

31개의 펩타이드 그룹이 잠재적인 생체활성을 가지고 있으며,

주로 항산화 특성, ACE 및 DPP-IV 억제 작용과 관련이 있는 것으로 나타났습니다.

루코노스토쿠스 슈도메센테로이데스(Leuconostoc pseudomesenteroides)로

만든 음료에서

생체활성 펩타이드의 양과 수가 가장 많았습니다.

이 연구는

잠재적인 건강 특성을 지닌

새로운 피스타치오 발효 음료를 설계하기 위한 기초를 마련합니다.

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Keywords

Fermentation

Pistachio beverage

Proteolysis

Lactic acid bacteria

Leuconostoc pseudomesenteroides

Companilactobacillus paralimentarius

Bioactive peptides

1. Introduction

Fermentation is a natural process practiced since ancient times to improve nutritional and sensory properties and food preservation (Ross and Morgan, 2022). Numerous scientific evidences have proven the high nutritional values and health benefits of fermented foods compared to the unfermented matrix, such as reduction blood cholesterol levels, increasing immune defences, preventing carcinogenesis, osteoporosis, diabetes, obesity, allergies, atherosclerosis, and alleviating symptoms in lactose-intolerant subjects (Şanlier et al., 2019).

Food fermentation is deeply embedded in local culture and traditions (Galimberti et al., 2021). Most traditional fermented foods are inducted by microorganisms spontaneously present in the starting raw material, resulting in a product of variable quality (Cuamatzin-García et al., 2022). Nowadays, modern food biotechnologies tend to optimize and standardize the fermentation process by employing selected starter microorganisms with well-defined characteristics, with the aim to improve flavor, safety and achieving high nutritional value and sustainability (Mannaa et al., 2021).

A wide variety of microbes can be responsible for the fermentation process, including lactic acid bacteria (LAB), acetic acid bacteria, yeasts and moulds (Campbell-Platt, 1994). In particular, LAB have been considered the most critical microbial group contributing to the beneficial effects of fermented foods/beverages (Ashaolu & Reale, 2020; de Souza et al., 2023; Kumar et al., 2022). LAB are able to ferment a variety of food substrates, such as milk, meat, fish, cereal, vegetables, and legumes (Ross and Morgan, 2002; Cuamatzin-García et al., 2022; Brown et al., 2017). Proteolytic events that occur during LAB fermentation lead to the release of bioactive peptides with potential health-promoting activities (Chai et al., 2020; Venegas-Ortega et al., 2019). Antioxidant, antihypertensive and antimicrobial peptides are the most remarkable subgroup of bioactive peptides that can be found in fermented foods (Martinez-Villaluenga et al., 2017, pp. 23–47).

Proteomics science has become an indispensable tool to examine proteolytic activity of microorganisms, providing insights on the function of fermenting microbiota in food products (Yang et al., 2020). The analysis of the peptidome and the dynamic changes during food fermentation and storage process, have been the subject of research over the years (Martini et al., 2021; Reale et al., 2021). Proteomic and peptidomic studies have been conducted to characterize and quantify the peptides released by proteolysis during LAB fermentation process (Dallas et al., 2015).

Dairy based products are popular fermented foods with beneficial effects on human health, and some of those benefits are related to protein-derived products (Widyastuti et al., 2021). Noteworthy, the consumption of dairy products is limited for a class of consumers suffering of lactose-intolerant, hypercholesterolemic or allergy, as well as for subjects adhering to vegan or vegetarian diets (Jaiswal & Worku, 2022). Vegetable foods/beverages have garnered significant attention as viable substitutes for dairy products (Mäkinen et al., 2016; Sethi et al., 2016). The impact of LAB on the nutritional properties over fermentation process of vegetable matrices has been previously investigated (Holscher, 2017; Kumar et al., 2022; Penha et al., 2020; Rekha & Vijayalakshmi, 2010; Zhao & Shah, 2014). Soybean and almond are the most used seeds to produce plant-based beverages, because of their technological properties. Peanuts, cashew, sesame, and rice are also employed to a lower extent (Harper et al., 2022).

Pistachio seeds are an important source of nutrients, including amino acids, dietary fibre, vitamins, and minerals. Clinical studies associated a diet including pistachios to a reduction in LDL levels and therefore lower risk of cardiovascular diseases and inflammatory bowel syndrome (Holligan, et al., 2014). In addition, the regular consumption of pistachio promotes, glycaemic control, appetite management, and weight control (Dreher, 2012). Pistachios are also a potentially suitable matrix for the fermentation process, as shown in previous studies (Sánchez-Bravo et al., 2020; Di Renzo et al., 2023) and the development of pistachio-based beverages, subject to additional fermentation, could enrich the range of plant-based beverages as an alternative to dairy–based products. In the present study, we used two starter cultures, Leuconostoc pseudomesenteroides D4 and Companilactobacillus paralimentarius G3, previously selected for their growth ability and acidifying activity in the pistachio matrix and for their impact on the odor profile of the model-beverage (Di Renzo et al., 2023). We performed a detailed eval‎uation of the proteolytic event occurring in the fermentation process of the pistachio beverage and the peptidome generated by the LAB was characterised using a proteomic approach with a focus on the identification of potentially bioactive peptides.

1. 서론

발효는 영양 및 감각적 특성 개선과 식품 보존을 위해 고대부터 행해져 온 자연적인 과정입니다(Ross and Morgan, 2022). 발효 식품의 높은 영양 가치와 건강상의 이점이 발효되지 않은 식품에 비해 우수하다는 사실은 수많은 과학적 증거를 통해 입증되었습니다.

예를 들어,

발효 식품은

혈중 콜레스테롤 수치를 낮추고,

면역력을 강화하며,

발암, 골다공증, 당뇨병, 비만, 알레르기, 동맥경화증, 유당 불내증 환자의 증상 완화에

도움이 된다는 사실이 밝혀졌습니다(Şanlier et al., 2019).

음식 발효는

지역 문화와 전통에 깊이 뿌리내리고 있습니다(Galimberti et al., 2021).

대부분의 전통적인 발효 식품은

원료에 자연적으로 존재하는 미생물에 의해 발효되어

품질이 다양한 제품이 됩니다(Cuamatzin-García et al., 2022).

오늘날, 현대 식품 생명공학은 잘 정의된 특성을 가진

선택된 스타터 미생물을 사용하여

발효 과정을 최적화하고 표준화하는 경향이 있으며,

풍미, 안전성, 높은 영양가 및 지속 가능성을 향상시키는 것을 목표로 합니다(Mannaa et al., 2021).

발효 과정에는

젖산균(LAB), 초산균, 효모, 곰팡이 등

다양한 미생물이 관여할 수 있습니다(Campbell-Platt, 1994).

특히,

LAB는 발효 식품/음료의 유익한 효과를 내는 데

가장 중요한 미생물 그룹으로 여겨져 왔습니다(Ashaolu & Reale, 2020; de Souza et al., 2023; Kumar et al., 2022).

LAB은

우유, 육류, 생선, 곡물, 채소, 콩류 등

다양한 음식물 기질을 발효할 수 있습니다(Ross and Morgan, 2002; Cuamatzin-García et al., 2022; Brown et al., 2017).

LAB 발효 과정에서 일어나는 단백질 분해 작용은

건강 증진에 도움이 되는

생체 활성 펩타이드의 방출을 유도합니다(Chai et al., 2020; Venegas-Ortega et al., 2019).

항산화, 항고혈압, 항균 펩티드는

발효 식품에서 발견되는

생체 활성 펩티드의 가장 주목할 만한 하위 그룹입니다(Martinez-Villaluenga et al., 2017, pp. 23–47).

프로테오믹스 과학은

미생물의 단백질 분해 활성을 조사하는 데 없어서는 안 될 도구로 자리 잡았으며,

식품에서 발효 미생물의 기능에 대한 통찰력을 제공합니다(Yang et al., 2020).

식품 발효 및 저장 과정 중

펩티돔과 동적 변화에 대한 분석은 수년 동안 연구의 주제였습니다(Martini et al., 2021; Reale et al., 2021).

LAB 발효 과정에서

단백질 분해에 의해 방출되는 펩티드를 특성화하고 정량화하기 위해

단백질 및 펩티드 연구가 수행되었습니다(Dallas et al., 2015).

유제품 기반 제품은

인체 건강에 유익한 효과가 있는 인기 있는 발효 식품이며,

이러한 효능 중 일부는 단백질 유래 제품과 관련이 있습니다(Widyastuti et al., 2021).

특히,

낙농 제품의 소비는

유당 불내증, 고콜레스테롤혈증 또는

알레르기로 고통받는 소비자층과 비건 또는 채식주의자(Jaiswal & Worku, 2022)에게

제한되어 있습니다.

식물성 식품/음료는

유제품을 대체할 수 있는 대안으로 주목받고 있습니다(Mäkinen et al., 2016; Sethi et al., 2016).

LAB가

식물성 매트릭스의 발효 과정에 미치는 영양적 영향에 대한 연구는

이미 진행되었습니다(Holscher, 2017; Kumar et al., 2022; Penha et al., 2020; Rekha & Vijayalakshmi, 2010; Zhao & Shah, 2014).

콩과 아몬드는

기술적 특성 때문에 식물성 음료를 생산하는 데 가장 많이 사용되는 씨앗입니다.

땅콩, 캐슈, 참깨, 쌀도

그보다 적은 양으로 사용됩니다(Harper et al., 2022).

피스타치오 씨앗은

아미노산, 식이섬유, 비타민, 미네랄을 포함한

중요한 영양소 공급원입니다.

임상 연구에 따르면 피스타치오를 포함한 식단은

LDL 수치를 감소시켜

심혈관 질환과 염증성 장 질환의 위험을 낮춘다고 합니다(Holligan, et al., 2014).

또한,

피스타치오를 규칙적으로 섭취하면

혈당 조절, 식욕 조절, 체중 조절에 도움이 된다고 합니다(Dreher, 2012).

피스타치오는 이전 연구(Sánchez-Bravo et al., 2020; Di Renzo et al., 2023)에서 알 수 있듯이

발효 과정에 적합한 매트릭스일 가능성이 있습니다.

피스타치오를 기반으로 한 음료를 개발하고

추가 발효 과정을 거치면

유제품 기반 제품에 대한 대안으로

식물성 음료의 종류를 다양화할 수 있습니다.

이 연구에서는

피스타치오 매트릭스에서 성장 능력과 산성화 활동,

그리고 모델 음료의 냄새 프로필에 미치는 영향으로 이전에 선택된

두 가지 스타터 배양균, Leuconostoc pseudomesenteroides D4와 Companilactobacillus paralimentarius G3를

사용했습니다(Di Renzo et al., 2023).

우리는

피스타치오 음료의 발효 과정에서 발생하는

단백질 분해 현상에 대한 상세한 평가를 수행했고,

잠재적인 생체 활성 펩타이드의 식별에 중점을 둔 단백질체학 접근법을 사용하여

LAB에 의해 생성된 펩티돔의 특성을 분석했습니다.

2. Material and methods

2.1. Chemicals

Trichloroacetic acid (TCA), trifluoroacetic acid (TFA), formic acid, acetonitrile, ammonium bicarbonate (AMBIC), cycloheximide, sodium chloride (NaCl), β-mercaptoethanol, Tris-HCl and Urea were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reagents for electrophoresis analysis were purchased from Bio-Rad (Segrate MI, Italy). Reagent for 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay was provided by Thermo Fischer Scientific (Bremem, Germany).

2.2. Strains and culture conditions

The LAB strains named Leuconostoc pseudomesenteroides D4 and Companilactobacillus paralimentarius G3 were previously identified and characterized for their tolerance to salinity, sucrose, ethanol, acid, and for proteolytic activity (Reale et al., 2020, 2021). Strains, belonged to the microbial culture collection of the Institute of Food Science - National Research Council (ISA-CNR, Avellino, Italy) were kept as frozen stocks (in 50% glycerol v/v) and routinely propagated in DeMan Rogosa and Sharpe (MRS) medium (Oxoid, Milan, Italy), pH 6.8 for 24 h at 30 °C.

Hereafter in the text, the LAB strains Leuconostoc pseudomesenteroides D4 and Companilactobacillus paralimentarius G3 will be referred to as D4 and G3, respectively.

2.3. Preparation of fermented pistachio beverage

Pistachio seeds (cv Bronte) were provided by a certified Italian producer of the Bronte variety (Aroma Sicilia, Bronte, Italy) produced in 2019 and store at 4 °C. De-hulled pistachio seeds were mixed with water (1:5 w:v) and ground in a colloidal mill (S.A.R. Group – model HOMO-Master 120) for about 5 min with recirculation, at room temperature. The resulting slurry was thermized at 70 °C for 30 min and cooled at 4 °C until the microbial inoculum. The inocula were prepared by sub-culturing each lactic acid bacteria strain in MRS broth incubated at 28 °C for 16 h, followed by centrifugation at 2000g for 10 min at 21 °C, washing of the pellets with sterile physiological solution (0.9% w/v NaCl) and final resuspension in the beverage at a final concentration of ∼6 logCFU/mL beverage. The beverages were fermented for 24 h at 28 °C and then stored at 4 °C for 30 days. In parallel, two control samples were prepared: a beverage without starter (control) and a beverage with 1% of lactic acid:acetic acid in ratio 4:1 v:v (acidified control). All beverage samples were collected after 24 h of incubation and stored at −80 °C until chemical-physical, microbiological and proteomic analysis.

2.3. 발효 피스타치오 음료의 준비
피스타치오 씨앗(품종 Bronte)은 2019년에 생산된 Bronte 품종(Aroma Sicilia, Bronte, Italy)의 이탈리아 인증 생산자가 제공했으며, 4°C에서 보관합니다. 

껍질을 벗긴 피스타치오 씨앗을 물(1:5 w:v)과 혼합하고, 

콜로이드 분쇄기(S.A.R. Group – 모델 HOMO-Master 120)에서 

약 5분 동안 재순환하면서 실온에서 분쇄했습니다. 

그 결과 생성된 슬러리를 

70°C에서 30분 동안 가열하고, 

미생물 접종원이 될 때까지 4°C에서 냉각했습니다. 

각 유산균 균주를 

28°C에서 16시간 동안 배양된 MRS 배지에서 서브 배양한 후, 

21°C에서 10분 동안 2000g으로 원심 분리하고, 

멸균 생리식염수(0.9% w/v NaCl)로 펠릿을 세척한 다음, 

음료에 최종 농도 약 6 log CFU/mL로 최종 재부유하여 접종액을 준비했습니다. 

음료는 

28°C에서 24시간 동안 발효시킨 다음 

4°C에서 30일 동안 보관했습니다. 

이와 동시에 두 개의 대조군 샘플을 준비했습니다: 스타터가 없는 음료(대조군)와 1%의 젖산:아세트산 비율 4:1 v:v(산성화 대조군). 모든 음료 샘플은 24시간 배양 후 수집되었으며, 화학-물리, 미생물학 및 단백질체학 분석이 이루어질 때까지 -80°C에서 보관되었습니다.

2.4. Determination of pH, acetic/lactic acid and LAB count

The pH value was determined using a BASIC 20 pH-meter (Crison Instruments, Barcelona, Spain) after diluting 1 mL of the beverage sample with 9 mL of distilled water, under magnetic stirring. For microbiological analysis, 1 mL of pistachio beverage was diluted with 9 mL of sterile physiological solution (0.9% w/v NaCl).

Acetic and lactic acid concentrations (expressed as g/L) were quantified using the RIDA®CUBE Assay Kits (Acetic acid RCS4226, D/L-Lactic Acid RCS4240, respectively) according to the manufacturer's instructions (R-Biopharm, Melegnano MI, Italy);.

LAB were counted on MRS (de Man, Rogosa and Sharpe) agar medium supplemented with 4 mg/100 mL of cycloheximide, after incubation at 28 °C for 72 h under anaerobic conditions (Gas Pack AnaeroGen TM, Oxoid, Milan, Italy).

Analyses of pH, LAB count, lactic and acetic acids were carried out in triplicate.

2.5. Degree of hydrolysis in fermented pistachio beverage

Proteolytic activity was determined by measuring the concentration of total primary amino groups (–NH2) by using the TNBS assay as reported by Adler-Nissen (1979). Briefly, 1 mL of freeze-dried pistachio beverage was solubilized in 0.5 M NaCl and 150 mM sodium phosphate at pH 6.8 to a ratio of 1:3 (w:v) and mixed for 30 min at 21 °C. After centrifugation at 12,000 g, for 20 min at 4 °C, 250 μL of supernatant was added to 250 μL of buffer borate and 500 μL of TNBS (%). Samples were incubated for 60 min at 37 °C. Reaction was stopped with 1 mL of phosphate buffer. Absorbance of the solution was measured spectrophotometrically at 420 nm using Ultrospec 160 2100 pro, (Amersham Bio-sciences, Uppsala, Sweden). The calibration curve was prepared using leucine (Leu) as standard in a range 0.0–1.0 mmol/L of Leu, and results were expressed as milligrams of Leu/L of beverage. The standard was assayed under reaction condition identical to those utilized for the samples. Experiments were performed in triplicate. TNBS analysis was performed using SYSTAT 13.0 for Windows (Systat Software Inc., Richmond, CA, USA).

2.6. SDS-PAGE analysis

The proteins of pistachio beverage (100 μL) were precipitated by adding an equal volume of acetone/TCA 20% and kept at −20 °C overnight. After centrifugation at 10,000 g for 10 min at 21 °C, the supernatant was discarded. Protein pellets were washed with 10 vol (three times) of pre-chilled acetone followed by centrifugation (10,000 g for 10 min at 21 °C). Pellets were dissolved in 20 μL of Laemmli buffer (0.125 M Tris−HCl pH 6.8, 5% SDS, 20% glycerol, 0.02% bromophenol blue) containing β-mercaptoethanol and heated in boiling water bath (100 °C) for 5 min. Proteins were separated by SDS-PAGE (Mini-Protean, Bio-Rad, Segrate MI, Italy) on a 12% polyacrylamide precast gels at 100 V. Gels were stained with Coomassie brilliant blue R-250 and destained in methanol:acetic acid:water(1:1:8) solution. The stained gels were imaged using GEL-DOCXR+ (Bio-Rad, Segrate MI, Italy).

2.7. LC–MS/MS analysis

Lyophilized pistachio beverage (1 mL) was suspended into 1 mL of urea 7 M Tris buffer saline (TBS) (50 mM Tris-HCl 150 mM NaCl) at pH 8 and incubated overnight at 4 °C under constant magnetic stirring. Sample was centrifuged at 10,000 g for 20 min at 21 °C, and the supernatant was collected. Proteins (MW > 7 kDa) and peptide (MW < 7 kDa) were fractionated by size exclusion chromatography (SEC), using an Econo-pack 10-DG pre-packed desalting column (Bio-Rad, Segrate MI, Italy), equilibrated, and eluted with 25 mM ammonium bicarbonate. The effluent was monitored by UV absorbance at 280 nm (Ultrospec 160 2100 pro, Amersham Biosciences, Uppsala, Sweden). The peptide-containing fractions were desalted using Sep-Pak C18 pre-packed cartridges (Waters, Milford, MA, USA), according to the manufacture's instruction and lyophilized. Peptide fraction was analyzed by LC–MS/MS using a Q Exactive Orbitrap mass spectrometer, equipped with a nano-electrospray ion source, and coupled online with an UltiMate 3000 RSLC nano system (Thermo Fischer Scientific, Bremem, Germany). Sample was suspended in 0.1% (v/v) TFA solution and transferred to polypropylene vials and nearly 1 μg was loaded through a 5 mm long, 300 mm i.d. pre-column and separated by an EASY-Spray™ PepMap C18 column (15 cm × 75 mm i.d.), 3 mm particles, 100 Å pore size (Thermo Fischer Scientific, Bremem, Germany). The separation was carried out with a linear gradient from 4% to 50% of 0.1% formic acid (v/v) in 80% acetonitrile (eluent B), over 50 min at a flow rate of 300 nL/min, after 5 min equilibration at 4% B. Eluent A was 0.1% formic acid (v/v) in Milli-Q water. The mass spectrometer was operated in data-dependent mode, and all MS1 spectra were acquired in the positive ionization mode in the mass scanning range 250–1600 m/z. Normalized collision energy was set to 27. The top-10 most intense precursor ions were selected for fragmentation in MS/MS mode, with 10 s of dynamic exclusion. A resolving power up to 70,000 full widths at half maximum (FWHM), an automatic gain control (AGC) target of 106 ions and a maximum ion injection time (IT) of 120 ms were set as standard values to generate precursor spectra. MS/MS fragmentation spectra were obtained at a resolving power of 17,500 FWHM. Spectra were elaborated using the Xcalibur Software version 3.1 (Thermo Fischer Scientific, Bremem, Germany).

2.8. Processing of LC-MS/MS data

Raw files were processed the Andromeda search engine of the MaxQuant open-source bioinformatic suite (https://maxquant.org). The searches were taxonomically restricted to the Pistacia vera databases (Taxon ID 55513) downloaded from UniprotKB. The search conditions included unspecific cleavage and no static modification. In both cases, methionine oxidation and pyroglutamic acid at N-terminus glutamine were selected as variable modifications. The mass tolerance value was 10 ppm for the precursor ion and 0.08 Da for MS/MS fragments. Peptide Spectrum Matches (PSMs) were filtered using the target decoy database approach with an e-value of 0.01 peptide-level false discovery rate (FDR), corresponding to a 99% confidence score. The average intensity of peptides was calculated based on the triplicate determination of the ion counts.

2.9. Bioinformatics analysis

Principal component analysis (PCA) was performed to show differential in mass peptide distribution among the fermented pistachio beverages using Perseus bioinformatics tools. Significant differences between means were determined by using one-way ANOVA followed by post hoc Bonferroni multiple comparison tests; a p-value <0.05 indicated statistical significance.

Potential bioactive peptides, released during fermentation, were in silico searched using as reference all peptides listed in the BIOPEP (http://www.uwm.edu.pl/biochemia) database (Iwaniak et al., 2016).

2.10. Statistical analysis

The statistical analysis was performed using the statistical software GraphPad Prism version 9.0 (GRAPH PAD software Inc, California, USA). Experiments were performed in triplicate and results were expressed as the mean ± standard deviation (SD). Significant differences (P < 0.05) were determined by analysis of one-way ANOVA, followed by Dunnett's test for multiple comparisons.

3. Results

3.1. Beverage preparation and characterization

Fig. 1 summarizes the analytical workflow applied to determine the enzymatic proteolysis in the inoculated, acidified and non-inoculated Pistachio beverages. All beverages were sampled at 24 h of fermentation for subsequent microbiology and physico-chemical and proteomics analyses.

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Fig. 1. Experimental workflow illustrating the technological and analytical strategy applied in this study for the assessment of pistachio fermented-beverages.

3.1.1. Microbiology and physico-chemical analysis

The pistachio-based beverage before inoculum had a pH of 6.45. In Table 1 are reported the results related to the pH, organic acid, microbial count and TNBS in the pistachio-based beverage after 24 h of fermentation.

Table 1. Eval‎uation of pH, microbial count, acidification and TNBS value in pistachio-based fermented beverages. Data are expressed as the mean ± SD of three experiments.

Beverage samplepHMRSlog CFU/mLLactic acid g/LAcetic acid g/LTNBS mg Leu eq./LControl (without microbial starter)Beverage chemically acidifiedBeverage fermented with D4Beverage fermented with G3

6.39 ± 0.01	<1.00	not detected	not detected	1.06 ± 0.08
3.95 ± 0.02	<1.00	4.91 ± 0.04	0.76 ± 0.06	1.20 ± 0.04
3.88 ± 0.08	9.30 ± 0.13	9.12 ± 0.03	0.43 ± 0.03	1.33 ± 0.10
3.91 ± 0.04	8.50 ± 0.15	7.87 ± 0.08	0.14 ± 0.04	1.38 ± 0.09

Both the strain starters (D4 and G3) were able to acidify the pistachio beverage. In fact, after 24 h of fermentation, the pH of LAB-fermented beverages decreased to 3.88 ± 0.08 (D4) and 3.91 ± 0.04 (G3), consistent with the production of lactic acid values, which ranged between 9.12 ± 0.03 (D4) and 7.87 ± 0.08 g/L (G3). The D4 and G3 starter cultures also induced production of acetic acid with a concentration of 0.43 ± 0.03 and 0.14 ± 0.04 g/L respectively. The low pH values of LAB-fermented beverages were comparable to the chemically acidified beverage, which had a value of about 3.95, which remained unchanged over the time. Similar, the pH and concentration of acetic acid and lactic acids of control beverage, did not change significantly over the 24 h incubation time.

As expected, the LAB counts of pistachio beverage at time 0 h were lower than 1.0 log CFU/mL, because of heat treatment of the beverage and LAB count was also negligible (1.0 log CFU/g) over the fermentation of control and acidified control beverages.

Furthermore, the pistachio matrix proved to be a suitable substrate for the growth of the strains. In fact, the cultures, inoculated at time zero with a value of about 6.0 log CFU/g, increased considerably reaching, after 24 h of fermentation, values of 9.30 ± 0.13 and 8.50 ± 0.15 log CFU/g for D4 and G3, respectively.

The proteolytic activity of LAB was determined by measuring the content of free amino groups in fermented pistachio beverages by TNBS assay. As shown in Fig. 2, the release of free amino groups was significantly higher in the beverages started by LAB G3 (1.38 ± 0.09 mg/L) and D4 (1.38 ± 0.10 mg/L) versus the control sample (1.06 ± 0.08 mg/L). Proteolysis degree over the 24 h was also observed for acidified beverages with a content of free-amino group of 1.20 ± 0.039 mg/L.

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Fig. 2. Degree of hydrolysis of pistachio-fermented beverages expressed as milligrams of Leucine/L of beverage. Each value is mean ± SD (n ≥ 3), and different superscripts (*; **) are significantly different at p < 0.05 and p < 0,01 respectively by Dunnett's multiple comparisons test.

3.1.2. Protein and peptide fraction analysis

As part of the analytical strategy outlined in Fig. 1, the proteomic analysis of sample beverages relied on the fractionation of protein fraction (MW > 7 kDa) from peptide fraction (MW < 7 kDa) by SE-chromatography. The protein fraction (MW > 7 kDa) was investigated by SDS-PAGE (Fig. 3). The electrophoretic patterns of control pistachio beverage ranged from 5 to 75 kDa corresponding to 2S albumin (9–15 kDa), 11S acid globulin subunit (20–30 kDa) and 11S basic globulin subunit (17–20 kDa), as previously reported (Di Stasio et al., 2022). No marked differences were observed in the electrophoretic profiles of control and acidified beverage samples. Conversely, LAB affected the SDS-PAGE profile due to their proteolytic activity. As a result of the fermentation process, LAB G3 and D4 beverage samples showed significant degradation of MW protein fractions 30–40 kDa which is accompanied by an increase in small polypeptides with molecular weight <20 kDa.

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Fig. 3. SDS-PAGE analysis of protein extract from pistachio beverages treated in different ways under reducing conditions. Lane M, Molecular marker (kDa); lane 1, protein of pistachio beverage (control); lane 2, proteins of acidified pistachio beverage; lane 3, proteins of fermented with D4 strain pistachio beverage; lane 4, proteins of fermented with G3 strain pistachio beverage.

The LC-MS/MS analysis of SEC-enriched peptide fraction, provided direct evidence of LAB-induced proteolytic activity after 24 h of fermentation. Peptide monitoring through LC-MS/MS, showed that the number and intensity of peptides arising from hydrolysis during 24 h of fermentation differed according to the G3 and D4 bacterial strain used to ferment the beverages with respect to control and acidified beverages (Fig. 4A). The detailed list of identified peptides in each beverage sample is shown in Supplementary Table 1. A total of 494 endogenous peptides were identified in the control (uninoculated) beverage. The chemical acidified beverage and those fermented with G4 and D4, had a number of peptides to 1065, 1178 and 1258 respectively. This result was in line with the increased amount of free-NH2 group as determined by TNBS assay. The Venn diagram in Fig. 4B illustrates the peptides found in chemical acidified beverage and those fermented with LAB. There were 661 coexisting peptides present in acidified and started samples. The microbial activity of the LAB determined the complexity of peptides, as 208 and 231 peptides specific peptides were found in the G3 and D4 samples, respectively. A total of 30 and 205 peptides in acidic beverages overlapped with G3 and D4, respectively, while 279 peptides overlapped between G3 and D4.

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Fig. 4. Peptidomics analysis of beverage samples. A full list of identified peptides is shown in Supplementary Table S1. Panel A: number of peptides derived from hydrolysis of pistachio proteins in G3-, D4-, acidified- and control beverages. Panel B: Venn diagrams of the number of peptides affected by proteolysis in G3-, D4- LAB strains and acidified control beverages. Panel C: Principal Component Analysis (PCA) scores plots of peptidome of G3-, D4-, acidified and control beverages; the contribution of the PC1 was 47,8%, while PC2 contributed for 20.4% of the total variance (when combined, PC1 and PC2 constituted 69,2% of the total variance). Panel D: mass distribution of the identified peptides of G3-, D4-, acidified and control beverages.

To examine the distinction between the peptide profiles of beverage samples, a principal component analysis (PCA) was performed considering the abundance of the identified peptides as loadings. As shown in Fig. 4C, the PCA analysis revealed that the replicates in each beverage sample were well clustered. The beverages inoculated with LAB and acidified beverages were significantly different from control beverage. The control sample clustered on its own, while G3 and D4 beverage samples clustered closely and distinctly from acidified sample.

Similarly, differences in peptide profiles were observed by comparison of the masses (Da) of the peptides identified by LC-MS/MS, as shown in Fig. 4D. The height of the bar (counts) indicates the ion abundance of peptides in that particular mass range. The LFQ proteomic analysis revealed an increased number of peptides ranging 1000–4500 Da in the samples fermented with LAB compared with both control samples. The mass ranges overlap between G3 and D4 fermented beverages but do not align perfectly with control and acidified control beverages. The number of counts of G3-and D4-fermented beverages was higher compared to unstarted samples.

3.1.3. Bioactive amino acid sequences

The peptides resulting from proteolysis were processed by BIOPEP database (Supplementary Table S2), that includes information about known bioactive peptides, in order to associate potential bioactivities to their sequences (Table 2). Database search was restricted to bioactive sequences longer or equal to 4, in order to increase the confidence of identification. A group of 31 bioactive peptides, mostly belonging to 11S globulin, was identified. These peptides were mainly associated with Angiotensin-Converting Enzyme (ACE) inhibition, antioxidative properties, dipeptidyl peptidase IV (DPP-IV) inhibition properties. In detail, samples G3, D4, acidified control and control (un-inoculated) contained 13, 20, 15 and 10 bioactive peptides, respectively (Table 2). The peptides harbouring sequences GVLY (BIOPEP ID 9325, ACE inhibitor), RALP (BIOPEP ID 9468–9469, ACE inhibitor and Renin inhibitor), LPILR (BIOPEP ID 9708-9716-8661, ACE inhibitor, antioxidant and hypotensive peptide), LPAGV (BIOPEP ID 9998-9997-9999, ACE inhibitor, antioxidant and renin inhibitor) and ILAP (BIOPEP ID 8647, DPP-IV inhibitor) were identified as the most representative in the pool of peptides generated during pistachio beverage fermentation. Fig. 5 focuses on the abundance of each fragment found in the different beverages. Fermented beverage D4 showed the highest number of identified peptides, the highest hydrolysis degree and notably, the larger abundance of bioactive precursor peptides. In general, the endogenous enzymatic activity under acidic conditions shows a large distribution of low abundant peptides characterised by “ragged ends”, while the enzymatic activity of D4-fermented beverage shows some characteristic dominant sequences. For example, the sequence VEK before the bioactive sequence GVLY is well preserved, while the N or F residues are always present respectively at the N- and C-terminus the sequence LPILR. Finally, the fragments containing the bioactive sequence RALP with high amount have characteristic dominating sequences (FRALPLDVIK, FRALPLDV, RALPLDV and FRALPL).

Table 2. List of potential bioactive peptides identified in pistachio beverages by LC-MS/MS and BIOPEP database. A detailed list of bioactive peptide search is shown in Supplementary Table S2.

3.1.3. 생체 활성 아미노산 서열

단백질 분해로 생성된 펩티드는 알려진 생체 활성 펩티드에 대한 정보를 포함하는 BIOPEP 데이터베이스(보충표 S2)를 통해 처리되어 잠재적인 생체 활성을 그 서열과 연관지었습니다(표 2). 데이터베이스 검색은 식별의 신뢰도를 높이기 위해 4개 이상의 생체 활성 서열로 제한되었습니다.

11S 글로불린에 주로 속하는

31개의 생체 활성 펩티드 그룹이 확인되었습니다.

이 펩티드는 주로

안지오텐신 전환 효소(ACE) 억제, 항산화 특성, 디펩티딜 펩티다제 IV(DPP-IV) 억제 특성과 관련이 있었습니다.

구체적으로, 샘플 G3, D4, 산성화된 대조군, 대조군(접종하지 않은 상태)에는

각각 13개, 20개, 15개, 10개의 생체 활성 펩티드가

포함되어 있었습니다(표 2).

펩티드는

GVLY(BIOPEP ID 9325, ACE 억제제),

RALP(BIOPEP ID 9468-9469, ACE 억제제 및 레닌 억제제),

LPILR(BIOPEP ID 9708-9716-8661, ACE 억제제, 항산화 및 저혈압 펩타이드),

LPAGV(BIOPEP ID 9998-9997-9999, ACE 억제제, 항산화 및 레닌 억제제) 및

ILAP(BIOPEP ID 8647, DPP-IV 억제제)는

피스타치오 음료 발효 과정에서 생성된 펩타이드 풀에서

가장 대표적인 것으로 확인되었습니다.

그림 5는 다양한 음료에서 발견된 각 조각의 풍부함에 초점을 맞추고 있습니다.

발효 음료 D4는

확인된 펩타이드의 수가 가장 많고,

가수분해 정도가 가장 높으며,

특히 생체 활성 전구체 펩타이드의 풍부도가 더 컸습니다.

일반적으로

산성 조건에서 내인성 효소 활성은

“끝이 뭉툭한” 특징을 가진 저농도 펩타이드의 큰 분포를 나타내는 반면,

D4 발효 음료의 효소 활성은 몇 가지 특징적인 우세한 서열을 나타냅니다.

예를 들어, 생체활성 서열 GVLY 앞에 VEK 서열이 잘 보존되어 있는 반면, 서열 LPILR의 N 또는 F 잔기는 항상 각각 N-말단과 C-말단에 존재합니다. 마지막으로, 생체활성 서열 RALP를 많이 포함하고 있는 단편들은 특징적인 지배적 서열(FRALPLDVIK, FRALPLDV, RALPLDV, FRALPL)을 가지고 있습니다.

표 2. 피스타치오 음료에서 LC-MS/MS와 BIOPEP 데이터베이스를 통해 확인된 잠재적 생체 활성 펩타이드 목록. 생체 활성 펩타이드 검색에 대한 자세한 목록은 보충 표 S2에 나와 있습니다.

ACE inhibithorBioactive peptide: GVLY (ID:9325)aLengthSequenceProtein family classificationMassControlAcidifiedD4G3

	8	SVEKGVLY	11S globulin Pis v 2.0201	893,48583	ND	ND	1E+09	ND
	9	SVEKGVLYQ	11S globulin Pis v 2.0201	1021,5444	ND	3,13E+08	ND	ND
	10	SVEKGVLYQN	11S globulin Pis v 2.0201	1135.5873	ND	4,57E+08	1E+09	5E+08
	10	VEKGVLYQNA	11S globulin Pis v 2.0201	1119,5924	ND	2,16E+08	7E+08	2E+08
ACE inhibithor/Renin inhibitor	Bioactive peptide: RALP (ID: 9468–9469) a
	Length	Sequence	Protein family classification	Mass	Control	Acidified	D4	G3
	7	RALPLDV	11S globulin Pis v 2.0101	782,46504	ND	ND	3E+08	ND
	8	FRALPLDV	11S globulin Pis v 2.0101	929,53345	2E+07	4,05E+08	9E+08	1E+08
	9	RVSVFRALP	11S globulin Pis v 2.0101	1043,624	ND	1,86E+08	9E+07	9E+07
	9	VFRALPLDV	11S globulin Pis v 2.0101	1028,6019	ND	13510005	ND	ND
	9	FRALPLDVI	11S globulin Pis v 2.0101	1042,6175	ND	26302331	6E+07	ND
	9	VSVFRALPL	11S globulin Pis v 2.0101	1000,607	ND	ND	2E+07	ND
	10	RVSVFRALPL	11S globulin Pis v 2.0101	1156,7081	ND	48889332	ND	1E+08
	10	FRALPLDVIK	11S globulin Pis v 2.0101	1170,7125	ND	2,41E+08	6E+08	ND
	10	RALPLDVIKN	11S globulin Pis v 2.0101	1137,687	ND	1,34E+08	ND	ND
ACE inhibitors/Antioxidative/Hypotensive peptide	Bioactive peptide: LPILR (ID: 9708–9716–8661) a
	Length	Sequence	Protein family classification	Mass	Control	Acidified	D4	G3
	6	LPILRF	11S globulin Pis v 2.0201	757,48505	ND	ND	2E+08	ND
	8	NLPILRF	11S globulin Pis v 2.0201	871,52797	3E+07	ND	ND	ND
	7	LNLPILR	11S globulin Pis v 2.0201	837,54362	1E+07	ND	2E+08	3E+07
	8	LNLPILRF	11S globulin Pis v 2.0201	984,61204	1E+08	ND	8E+08	2E+08
	8	ALNLPILR	11S globulin Pis v 2.0201	908,58074	1E+07	ND	4E+08	7E+07
	8	NLPILRFL	11S globulin Pis v 2.0201	984,61204	2E+07	ND	ND	ND
	9	ALNLPILRF	11S globulin Pis v 2.0201	1055,6492	5E+07	2,45E+08	2E+09	4E+08
	9	LNLPILRFL	11S globulin Pis v 2.0201	1097,6961	3E+07	ND	ND	ND
	10	NLPILRFLQL	11S globulin Pis v 2.0201	1225,7547	ND	ND	2E+07	2E+07
	10	NALNLPILRF	11S globulin Pis v 2.0201	1169,6921	1E+07	ND	ND	4E+06
	10	ALNLPILRFL	11S globulin Pis v 2.0201	1168,7332	1E+07	ND	ND	ND
ACE inhibitors/Antioxidative/Renin inhibitor	Bioactive peptide: LPAGV (ID: 9998–9997–9999) a
	Length	Sequence	Protein family classification	Mass	Control	Acidified	D4	G3
	8	LPAGVAHW	11S globulin Pis v 2.0101	849,44972	ND	3,62E+08	ND	ND
	9	ALPAGVAHW	11S globulin Pis v 2.0101	920,48684	ND	ND	3E+08	ND
	9	VIALPAGVA	11S globulin Pis v 2.0101	809,50109	ND	11999267	ND	ND
	10	IALPAGVAHW	11S globulin Pis v 2.0101	1033,5709	ND	2,73E+09	3E+09	4E+08
	10	VIALPAGVAH	11S globulin Pis v 2.0101	946,56	ND	ND	4E+08	ND
DPP-IV inhibitor	Bioactive peptide: ILAP (ID:8647) a
	Length	Sequence	Protein family classification	Mass	Control	Acidified	D4	G3
	8	AILAPHWN	11S globulin Pis v 2.0101	920,48684	ND	ND	3E+08	3E+07
	9	RDAILAPHW	11S globulin Pis v 2.0101	1077,572	ND	98612007	5E+07	ND

a

ID number of bioactive sequence annotated in BIOPEP database.

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Fig. 5. Graphical representation of the relative abundance of precursor peptides (from 3 up to 10 amino acid residues) of A) the angiotensin-converting enzyme (ACE) inhibitor sequence RALP (BIOPEP ID 9468) and GVLY (BIOPEP ID 9325) B) the ACE inhibitor (BIOPEP ID 9708) and antioxidative (BIOPEP ID 9716) sequence LPILR and the ACE inhibitor (BIOPEP ID 9998) and antioxidative (BIOPEP ID 9997) sequence LPAGV and C) the dipeptidyl peptidase IV inhibitor (BIOPEP ID8647) sequence ILAP.

그림 5. A) 안지오텐신 전환 효소(ACE) 억제제 서열 RALP(BIOPEP ID 9468)와 GVLY(BIOPEP ID 9325)의 전구체 펩티드(3~10개의 아미노산 잔기)의 상대적 풍부도를 그래픽으로 표현한 그림 B) ACE 억제제(BIOPEP ID 9708) 및 항산화제(BIOPEP ID 9716) 서열 LPILR 및 ACE 억제제(BIOPEP ID 9998) 및 항산화제(BIOPEP ID 9997) 서열 LPAGV 및 C) 디펩티딜 펩티다제 IV 억제제(BIOPEP ID8647) 서열 ILAP.

4. Discussion

In this study, we applied proteomics analysis to investigate the release of peptides from pistachio proteins by LAB fermentation of pistachio-based beverage. Certain LAB have a complex proteolytic system that includes proteases to hydrolyze food proteins, transport systems to incorporate these peptides, and peptidase to metabolize peptides into amino acids and nitrogen essential for survival (Savijoki et al., 2006). However, it is documented in different food system that acidification induced by LAB clearly trigger proteolysis through activation of flour endogenous proteases (Gänzle et al., 2008). Previously studies on cereals, for example, have described a comparable protein degradation between chemically acidified matrix and LAB started fermented matrix (Gänzle, 2014; Thiele et al., 2002).

For this reason, in this study, in order to establish the role of endogenous pistachio proteases and LAB proteases, a control acidified beverage was analyzed in parallel. According to our findings, the peptides accumulated in the fermented beverage resulted from both the activity of endogenous pistachio proteases activated at low pH and the activity of LAB proteases. Undoubtedly, the acidification of the medium, caused the activation of the endogenous proteases, as demonstrated by the results of the proteolytic data. It should also be noted that the heat treatment at 70 °C carried out on the drink, was useful in reducing adventitious microorganisms (in fact, the charges were <1 log CFU/mL after treatment), but not adequate in denaturing the endogenous proteases that were still active. Furthermore, although LAB G3 and D4 acidified the beverage to similar pH values, the proteolytic patterns differed from each other and both from the acidified control. The number and frequency of identified peptides were higher in the beverage fermented with D4, followed by G3 and chemically acidified beverage. These results suggest that, in addition to endogenous protease active at acidic pH, the proteolytic system of LAB directly participated in peptide degradation to some extent, in line with previous findings (Reale et al., 2021; Spiecher & Nierle, 1988). After 24 h of LAB fermentation, 2S albumin, 7S and 11S globulins were the main pistachio proteins almost completely hydrolyzed. These proteins are storage proteins involved in seeds allergy. Proteolysis has denatured the 2S albumin, 7S and 11S globulins, thereby reducing their IgE-binding capacity and, potentially, their allergenicity (Gänzle, 2014).

Another important result is that fermentation leads to the production of peptides with potential bioactive properties. Overall, the peptides present in the fermented beverage varied significantly, with D4 containing the most peptides, followed by G3. This could be attributed to variations in enzymatic specificity between the different strains. In fact, LAB cell-envelope proteinases vary in substrate specificity, domain composition and anchoring mechanism, factors that may influence the production of protein hydrolysate (Savijoki et al., 2006). Using a predictive informatics tool based on the BIOPEP database, the peptides identified in fermented beverages showed a high frequency of ACE inhibitory, antioxidant and DPP-IV inhibitors sequences. In general, bioactive peptides contain mainly 3–20 amino acid units, but in some cases the size is larger (Shahidi & Zhong, 2008), and can be considered as components of functional foods which may exert regulatory activities in the human organism, irrespective of their nutritive functions (Gobbetti et al., 2007). Sample D4 had the highest content of ACE inhibitory peptides with antioxidant and DPP-IV inhibitor activity compared to G3. Interest in ACE-inhibitory peptides has grown as it has been shown that they can be competitive substrates for the inhibition of angiotensin I-converting enzyme, which plays a key role in the regulation of blood pressure (Iwaniak et al., 2014). Previous studies have report the identification of ACE inhibitory peptides from fermented plant food (Ambigaipalan et al., 2015; Guang & Phillips, 2009; Ramlal et al., 2022). Similarly, biologically active peptides with potential antioxidant activity have been isolated from plant fermented food matrices. (Rizzello et al., 2016; García et al., 2013; Babini et al., 2017). The antioxidant potential of bioactive peptides is based on their capacity to transfer hydrogen or electrons, preventing oxidative stress associated with numerous degenerative diseases like cancer and atherosclerosis (Chakrabarti and Jahandideh, 2014; Coda et al., 2012). Although the antioxidant properties of fermented foods have mostly been attributed to the presence of phenolic compounds (Chakrabarti and Jahandideh, 2014), the release of antioxidant peptides during fermentation may significantly contribute to the bioactivity of the final product (García et al., 2013). Interest in peptides with DPP-IV inhibitor activity has been discovered in various fermented foods. Dipeptidyl peptidase-IV inhibitor is a peptide that can increase insulin secretion and, therefore, decrease blood glycaemia by preventing incretins inactivation (Zhang et al., 2022).

4. 토론

이 연구에서는 피스타치오 기반 음료를 발효시킨 락토바실러스(LAB)가 피스타치오 단백질에서 펩티드를 방출하는 것을 조사하기 위해 단백질체학 분석을 적용했습니다.

특정 락토바실러스는

식품 단백질을 가수분해하는 프로테아제,

이러한 펩티드를 통합하는 수송 시스템,

생존에 필수적인 아미노산과 질소로 펩티드를 대사하는 펩티다제를 포함하는

복잡한 단백질 분해 시스템을 가지고 있습니다(Savijoki et al., 2006).

그러나,

다른 식품 시스템에서 LAB에 의해 유발된 산성화가

밀가루 내인성 프로테아제의 활성화를 통해

단백질 분해를 명확하게 유발한다는 것이 문서화되어 있습니다(Gänzle et al., 2008).

예를 들어, 곡물에 대한 이전 연구에서는

화학적으로 산성화된 매트릭스와 LAB에 의해 발효된 매트릭스 사이에

유사한 단백질 분해 현상이 발생한다고 설명했습니다(Gänzle, 2014; Thiele et al., 2002).

이러한 이유로, 이 연구에서는 내인성 피스타치오 프로테아제와 락토바실러스 프로테아제의 역할을 규명하기 위해, 대조군인 산성화 음료를 동시에 분석했습니다.

연구 결과에 따르면,

발효 음료에 축적된 펩타이드는

낮은 pH에서 활성화된 내인성 피스타치오 프로테아제와

락토바실러스 프로테아제의 활성에 의해 생성된 것입니다.

의심할 여지 없이,

산성화된 환경은 내인성 프로테아제의 활성화를 유발했으며,

이는 단백질 분해 데이터의 결과로 입증되었습니다.

또한

음료에 70°C의 열처리를 실시하면

우발적 미생물을 줄이는 데 도움이 된다는 점도 주목할 만합니다

(실제로 처리 후 부하는 1 log CFU/mL 미만).

그러나

여전히 활성화된 내인성 프로테아제를 변성시키기에 충분하지는 않습니다.

또한,

LAB G3와 D4가 음료를 비슷한 pH 값으로 산성화시켰지만,

단백질 분해 패턴은 서로 달랐고,

산성화된 대조군과도 달랐습니다.

확인된 펩티드의 수와 빈도는 D4로 발효된 음료에서 더 높았고,

그 다음으로 G3와 화학적으로 산성화된 음료 순이었습니다.

이러한 결과는

산성 pH에서 활성화되는 내인성 프로테아제 외에도,

LAB의 단백질 분해 시스템이

어느 정도 펩티드 분해에 직접 관여했음을 시사합니다.

이는 이전 연구 결과(Reale et al., 2021; Spiecher & Nierle, 1988)와 일치합니다.

LAB 발효 24시간 후,

2S 알부민,

7S 및 11S 글로불린이 거의 완전히 가수분해되어

피스타치오 단백질의 주성분이 되었습니다.

이 단백질들은

씨앗 알레르기와 관련된 저장 단백질입니다.

단백질 분해는

2S 알부민, 7S 및 11S 글로불린의 변성을 일으켜,

이로 인해 이들의 IgE 결합 능력과

잠재적인 알레르기 유발 가능성이 감소되었습니다(Gänzle, 2014).

또 다른 중요한 결과는

발효가 잠재적인 생체 활성 특성을 가진

펩티드의 생산으로 이어진다는 것입니다.

전반적으로 발효 음료에 존재하는 펩티드는

D4가 가장 많은 펩티드를 함유하고 있으며,

그 뒤를 G3가 따르는 등 매우 다양했습니다.

이는 다른 균주들 사이의 효소 특이성의 차이에 기인할 수 있습니다.

실제로,

LAB 세포 외막 단백질 분해효소는

단백질 가수분해물의 생산에 영향을 미칠 수 있는 기질 특이성,

도메인 구성 및 고정 메커니즘의 측면에서 차이가 있습니다(Savijoki et al., 2006).

BIOPEP 데이터베이스를 기반으로 하는 예측 정보학 도구를 사용하여

발효 음료에서 확인된 펩티드는

ACE 억제, 항산화 및 DPP-IV 억제 서열의 높은 빈도를 보였습니다.

일반적으로

생체 활성 펩티드는

주로 3~20개의 아미노산 단위로 구성되어 있지만,

경우에 따라 그 크기가 더 클 수 있습니다(Shahidi & Zhong, 2008).

그리고

영양 기능과 관계없이

인체에서 조절 작용을 발휘할 수 있는 기능성 식품의 구성 요소로 간주될 수 있습니다(Gobbetti et al., 2007).

샘플 D4는 G3에 비해 항산화 및 DPP-IV 억제 활성을 가진 ACE 억제 펩타이드의 함량이 가장 높았습니다.

ACE 억제 펩타이드에 대한 관심은

혈압 조절에 중요한 역할을 하는 안지오텐신 I 전환 효소의 억제에

경쟁적인 기질일 수 있다는 사실이 밝혀지면서 증가했습니다(Iwaniak et al., 2014).

이전 연구에서는

발효 식물성 식품에서

ACE 억제 펩타이드를 확인했다고 보고했습니다(Ambigaipalan et al., 2015; Guang & Phillips, 2009; Ramlal et al., 2022).

마찬가지로,

식물성 발효 식품 매트릭스에서 잠재적인 항산화 활성을 가진

생물학적 활성 펩타이드가 분리되었습니다. (Rizzello et al., 2016; García et al., 2013; Babini et al., 2017).

생체 활성 펩티드의 항산화 잠재력은

수소 또는 전자를 전달하는 능력에 기반을 두고 있으며,

암과 죽상 동맥 경화증과 같은 수많은 퇴행성 질환과 관련된

산화 스트레스를 방지합니다(Chakrabarti and Jahandideh, 2014; Coda et al., 2012).

발효 식품의 항산화 특성은

주로 페놀 화합물의 존재에 기인한다고 알려져 있지만(Chakrabarti and Jahandideh, 2014),

발효 과정에서 항산화 펩타이드가 방출되면

최종 제품의 생체 활성에 크게 기여할 수 있습니다(García et al., 2013).

DPP-IV 억제 활성을 가진 펩타이드에 대한 관심이

다양한 발효 식품에서 발견되었습니다.

디펩티딜 펩티다제-IV 억제제는

인슐린 분비를 증가시켜 인크레틴의 비활성화를 방지함으로써

혈당 수치를 감소시킬 수 있는 펩타이드입니다(Zhang et al., 2022).

5. Conclusions

In conclusion, this research has shed light on the potential of fermented-pistachio beverage with probiotic, nutritional and health benefits. A variety of peptides with different sequences and lengths are generated in beverage samples as a consequence of proteolysis induced by LAB. Some of the released peptides own potentially bioactivity like inhibition of ACE, DPP-IV and antioxidant properties. Results also showed that protein degradation is mainly affected by endogenous protease naturally occurred in pistachio seed, but LAB proteases affecting the degradation of proteins during fermentation also increase the concentration and the patterns of peptides. However, to fully harness benefits of pistachio based-beverage for consumers, further studies are necessary to demonstrate the effectiveness of the health positive activities suggested by the present in silico data. Synthesis of the selected predicted peptides followed by in vitro and in vivo eval‎uation may allow to confirm‎ the peptide bioactivity. At the same time, stimulated gastrointestinal conditions and cell culture mimicking the intestinal absorptive environment is needed to fully understand the bioaccessibility and bioavailability of these fermentation-derived peptides.

5. 결론

결론적으로, 이 연구는 발효 피스타치오 음료가 가진 프로바이오틱, 영양, 건강상의 이점에 대한 잠재력을 밝혀냈습니다. LAB에 의해 유도된 단백질 분해의 결과로, 음료 샘플에서 서로 다른 서열과 길이를 가진 다양한 펩타이드가 생성됩니다.

방출된 펩타이드 중 일부는

ACE, DPP-IV 억제 및 항산화 특성과 같은

잠재적인 생체 활성을 가지고 있습니다.

또한, 피스타치오 씨앗에 자연적으로 존재하는

내인성 프로테아제가

단백질 분해에 주로 영향을 미치지만,

발효 과정에서 단백질 분해에 영향을 미치는

LAB 프로테아제도 펩타이드의 농도와 패턴을 증가시킨다는 결과가 나왔습니다.

그러나 소비자가 피스타치오 음료를 최대한 활용할 수 있도록 하기 위해서는, 현재의 컴퓨터 시뮬레이션 데이터가 제시하는 건강에 긍정적인 활동의 효과를 입증하기 위한 추가 연구가 필요합니다. 선택된 예측 펩타이드의 합성과 그 후의 체외 및 체내 평가를 통해 펩타이드의 생체 활성을 확인할 수 있습니다. 동시에, 이러한 발효 유래 펩타이드의 생체 접근성과 생체 이용률을 완전히 이해하기 위해서는 자극된 위장 상태와 장내 흡수 환경을 모방한 세포 배양이 필요합니다.

CRediT authorship contribution statement

Serena Marulo: Writing – original draft, Formal analysis, Data curation. Salvatore De Caro: Formal analysis, Data curation, Conceptualization. Chiara Nitride: Formal analysis, Data curation, Conceptualization. Tiziana Di Renzo: Data curation, Conceptualization. Luigia Di Stasio: Writing – review & editing, Visualization, Data curation. Pasquale Ferranti: Writing – original draft, Data curation, Conceptualization. Anna Reale: Writing – review & editing, Funding acquisition, Formal analysis, Data curation, Conceptualization. Gianfranco Mamone: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of Competing interest

The authors confirm‎ that they have no conflicts of interest with respect to the work described in this manuscript.

Acknowledgements

This research was funded by the Bilateral project between ISA-CNR e Centro de Investigacion y Desarrollo en Criotecnología de Alimentos (CONICET- UNLP-CIC Buenos Aires) entitled “Development of functional pistachio-based fermented beverage” (SAC.AD002.001.024) and by the project NUTRAGE FOE-2021 DBA.AD005.225.

We, are grateful to Angela Sorrentino (Centre for Food Innovation and Development of the Food Industry, University of Naples Federico II), for her technical support in the pistachio grinding process with the colloidal mill.

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References

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